<p class="jove_content">The authors would like to thank Kevin Graham for his voice-over and all participants who donated samples for this work.</p>

<p class="jove_content">NOTE: This protocol is only to be used if students or respective legal guardians agreed to the conductance of this protocol as genotypic profiles give insights into genetic relationships. DNA fingerprinting is a common molecular biology method mainly applied to population studies and forensic matters. Therefore, attention should be devoted to keep contamination risks as low as possible. To avoid contamination of the sample with DNA from an outside source or DNases, gloves should be worn, instruments should be thoroughly cleaned or sterilized and solutions should be filter-sterilized or autoclaved before usage.</p>
<p class="jove_title">1. Harvesting of buccal mucosa epithelial cells</p>
<p class="jove_content">CAUTION: Work with saliva and epithelial cells can lead to a transmission of infectious diseases. Therefore, standard and transmission-based precautions should be applied (e.g., the use of appropriate personal protective equipment).</p>
<p class="jove_content">NOTE: Include a positive control, which is provided by the instructor (e.g., DNA extracted by the instructor) and included in the downstream processing steps.</p>
<ol>
	<li>Wait at least 1 h after eating or brushing teeth before specimen collection.</li>
	<li>Label an empty and sterile 2.0 mL microcentrifuge tube.</li>
	<li>Remove one sterile buccal swab from the packaging and rub vigorously on the inside of the cheek for 30-40 times or 30-40 seconds to harvest buccal mucosa epithelial cells.</li>
	<li>Place the tip of the collection swab into the previously labeled sterile microcentrifuge tube and break off the length of plastic that extends beyond the edge, either by hand or using sterile scissors.</li>
	<li>Place the cap securely onto the tube, sealing the collection swab inside.</li>
</ol>
<p class="jove_title">2. Extraction of genomic DNA from human cells</p>
<ol>
	<li>Before extraction, set a thermal mixer or a heating block to 65 &#176;C for sample lysis.<br />
	CAUTION: Some chemicals used are classified hazardous. Read the safety data sheet carefully and take appropriate safety measures before handling.
	<ol>
		<li>Prepare and sterilize by filtering or autoclaving all required buffers and solutions (lysis solution, 8 M potassium acetate, 2-propanol, 70% ethanol and elution buffer).<br />
		NOTE: All centrifugation steps should be performed at room temperature (20-30 &#176;C) unless specified.</li>
	</ol>
	</li>
	<li>Add 500 &#956;L of lysis solution (50 mM Tris/HCl, pH 8.0; 10 mM ethylenediamine tetra-acetic acid (EDTA); 2% sodium dodecyl sulfate (SDS)) to the buccal swab, making sure that the sample is completely immersed in the lysis solution.
	<ol>
		<li>Vortex vigorously for at least 5 s.</li>
		<li>Incubate samples at 65 &#176;C in a thermal mixer for 10 min.</li>
		<li>Mix sample 3-4 times by pulse-vortexing for 5 s during incubation.</li>
		<li>Remove swab from the lysis buffer, press the swab against the inside of the tube to obtain maximum sample volume.</li>
	</ol>
	</li>
	<li>Add 100 &#956;L of 8 M potassium acetate to lysed cells.
	<ol>
		<li>Mix thoroughly by inverting the tube until there is a white precipitate.</li>
		<li>Incubate for 5 min at room temperature.</li>
	</ol>
	</li>
	<li>Centrifuge the sample for 5 min at 18,000 <em>x g</em>.</li>
	<li>Transfer 450 &#956;L of the supernatant into a clean and sterile 1.5 mL microcentrifuge tube.</li>
	<li>Add 450 &#956;L of 2-propanol and mix thoroughly by inverting the tube (precipitation of the DNA).</li>
	<li>Incubate for 5 min at room temperature.</li>
	<li>Centrifuge for 5 min at 18,000 <em>x g</em>.</li>
	<li>Discard the supernatant and invert the tube on a clean paper towel to dry the pellet and to avoid cross-contamination.</li>
	<li>Incubate the DNA for 5 min at 65 &#176;C in a heating block to dry the pellet completely.</li>
	<li>To wash the DNA, add 500 &#956;L of 70% ethanol.</li>
	<li>Centrifuge for 1 min at 18,000 <em>x g</em>.</li>
	<li>Discard the supernatant and invert the tube on a clean paper towel to dry the pellet.</li>
	<li>Add 30 &#956;L of resuspension buffer (10 mM Tris/HCl pH 8.0, 1 mM EDTA) to the pellet.</li>
	<li>Incubate the DNA for 10 min at 65 &#176;C in a heating block to inactivate DNases.</li>
</ol>
<p class="jove_title">3. Amplifying the <em>D1S80</em> VNTR locus using PCR</p>
<p class="jove_content">NOTE: Record the number of samples that will be used and prepare a worksheet with the required reagents and their volumes before collecting the necessary plastics and other materials. Label the sterile tubes/strips/plates to be used for the PCR with sample numbers. Remember to include a negative control using H<sub>2</sub>O instead of DNA and a positive control (e.g.,&#160;using the DNA provided by the instructor) to validate the PCR.</p>
<ol>
	<li>Prepare the 1x PCR master mix containing 10 &#956;L of 5x PCR reaction buffer (containing 15 mM MgCl<sub>2</sub>), 1 &#956;L of deoxyribonucleotide triphosphate (dNTPs) (10 mM), 5 &#956;L (10 pmol) of each primer pMCT118-f and pMCT118-r (forward - 5&#39;-GAAACTGGCCTCCAAACACTGCCCGCCG-3&#39;, reverse - 5&#39;-GTCTTGTTGGAGATGCACGTGCCCCTTGC-3&#39;) according to Kasai et al.<sup class="xref">2</sup>, 23.8 &#956;L of ultrapure H<sub>2</sub>O, and 0.2 &#956;L of Taq DNA polymerase (5 U/&#181;L).<br />
	NOTE: The primers pMCT118-f/pMCT118-r were designed on the flanking regions of the <em>D1S80</em> VNTR region amplifying the whole locus.</li>
	<li>Label the PCR tubes/strips/plate with the sample numbers to be used.</li>
	<li>Aliquot 45 &#956;L of the master mix to each labeled PCR sample tube or well.</li>
	<li>Add 5 &#956;L of the DNA template to the master mix in each PCR tube/plate well to acquire a total volume of 50 &#181;L. Change the pipette tip for every DNA sample to avoid cross contamination.</li>
	<li>Include a no template control (NTC) by using ultrapure H<sub>2</sub>O instead of DNA.</li>
	<li>Close PCR tubes/strips or seal plate and mix.</li>
	<li>Centrifuge the PCR tubes/strips/plates for 20 s using a tabletop centrifuge.<br />
	NOTE: PCR conditions given in this protocol were optimized using the DNA polymerase and the PCR thermal cycler used. In general, PCR conditions must be adapted to the DNA polymerase. The standard extension time for a Taq DNA polymerase is 1 min/kb.</li>
	<li>Place the sample tubes/strips/plate in the thermocycler and incubate reactions using the following conditions (DNA polymerase extension time): 1 cycle of 95 &#176;C for 2 min; 25 cycles of 94 &#176;C for 15 s, 60 &#176;C for 15 s, 72 &#176;C for 30 s; 1 cycle of 72 &#176;C for 10 min.</li>
	<li>When the program is complete, remove the products from the thermocycler and store at 4 &#176;C overnight or -20 &#176;C until electrophoresis.</li>
</ol>
<p class="jove_title">4. Agarose gel electrophoresis of PCR products</p>
<ol>
	<li>Prepare a 1.5% agarose gel.
	<ol>
		<li>Use 1.5 g of agarose powder and mix it with 100 mL of 1x Tris-acetate-EDTA (TAE) buffer solution in a flask.</li>
		<li>Heat the mixture for about 1.5-2 min in a microwave oven (600 W). Swirl the contents and heat again, if necessary, to completely dissolve the agarose. Cool slightly and add 2 &#956;L of PeqGreen to the agarose.</li>
		<li>Pour the gel in a form using a comb with enough wells for all samples and add at least one molecular weight marker.<br />
		CAUTION: Boiling retardation may occur. Shake the flask carefully when resuspending the agarose which has not been dissolved yet.<br />
		NOTE: PeqGreen is a non-toxic dye for the detection of nucleic acids. It is applicable for staining of double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) as well as RNA. The sensitivity is comparable to ethidium bromide.</li>
	</ol>
	</li>
	<li>Remove the PCR products from 4 &#176;C and centrifuge for about 10 s.</li>
	<li>Load the wells of the gel with 10 &#956;L sample. Do not overload the gel.<br />
	NOTE: The DNA polymerase buffer also includes compounds that increases sample density so that samples can be loaded directly onto gels without the need for a loading dye. This allows the sample to sink into the well and dyes help to track how far the DNA sample has migrated.
	<ol>
		<li>Add a molecular weight standard to the flanking wells (preferably a 50 bp molecular weight standard).<br />
		NOTE: Store the residual PCR samples at -20 &#176;C in case the agarose gel electrophoresis has to be repeated.</li>
	</ol>
	</li>
	<li>Run the gel in 1x TAE running buffer at 150 V (constant) for approximately 40 min or until the lower yellow dye front that travels at around 50 bp reaches the lower end of the gel.</li>
	<li>Image the gel while blue light or ultraviolet (UV) light is applied and record an image for fragment length analysis.<br />
	CAUTION: UV light can damage your eyes and skin. Always wear protective clothing and UV safety glasses when using a UV light box.</li>
	<li>Dispose the gel in accordance with the institutional hazardous materials policy.</li>
</ol>
<p class="jove_title">5. Analysis of fragment length results</p>
<p class="jove_content">NOTE: Use linear regression analysis to estimate the lengths of the fragments.</p>
<ol>
	<li>For sizing the <em>D1S80</em> PCR fragments, place a ruler on the gel photograph over the 50 bp molecular weight standard lane such that the top of the ruler lines up with the bottom of the well into which the sample was loaded (<strong class="xfig">Figure 1</strong>).<br />
	NOTE: To calculate the linear regression equation for the molecular weight standard (50 bp molecular weight standard) a spreadsheet program was used.</li>
	<li>Record the distance from the well (e.g., in cm) for each band of the 50 bp molecular weight standard in a table using a spreadsheet program (<strong class="xfig">Figure 2</strong>).</li>
	<li>Determine the log (e.g., base 10) of each fragment size of the 50 bp molecular weight standard and enter the log values into the table (<strong class="xfig">Figure 2</strong>).</li>
	<li>Plot each data point to a graph with the log of the band sizes on the vertical axis (y-axis) and the measured run distance from the top of the gel to each band of the molecular weight standard on the horizontal axis (x-axis) using a scatterplot (<strong class="xfig">Figure 3</strong>).</li>
	<li>Fit a trendline (linear regression line) and show the regression equation (y = ax + b) and the R<sup>2</sup> value on the graph (<strong class="xfig">Figure 4</strong>).</li>
	<li>Measure the distance migrated (e.g., in cm) for each <em>D1S80</em> amplicon&#160;(<strong class="xfig">Figure 5</strong>).</li>
	<li>Estimate the size of each <em>D1S80</em> amplicon&#160;using the regression equation: y = ax + b<br />
	where y = the log of the fragment size<br />
	&#160; &#160; &#160; &#160; &#160; &#160;a = the slope of the line (calculated in point 5)<br />
	&#160; &#160; &#160; &#160; &#160; &#160;x = the distance from the well (in cm)<br />
	&#160; &#160; &#160; &#160; &#160; &#160;b = the point where the regression line intercepts the y-axis (calculated in point 5)<br />
	&#8203;NOTE: Since the y-value represents the log of the fragment size, the antilog (10<sup>y</sup>) must be calculated to obtain the fragment size in bp of the <em>D1S80</em> amplicons.</li>
</ol>
<p class="jove_title">6. Estimation of the number of repeat units in the alleles of the tested individuals</p>
<p class="jove_content">NOTE: The <em>D1S80</em> repeat unit is 16 bp in length. The smallest known allele for <em>D1S80</em> has 14 repeats. The amplicon scheme described here produces amplicons with flanking regions adding up an extra 145 bp to the final size.</p>
<ol>
	<li>Use <strong>Table 1</strong> to estimate how many repeat units are contained in each PCR fragment. The size extrapolated using the linear regression should be within 8 bp of any particular allele.</li>
	<li>Record the genotype of each tested individual as a combination of allele repeat size numbers.</li>
</ol>

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H., Peacock, K., Dunning, A. M., Talmud, P., Humphries, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+relationship+between+the+3+'-VNTR+and+diallelic+apolipoprotein+B+gene+polymorphisms:+Haplotype+analysis+in+individuals+of+European+and+South+Asian+origin.">Genetic relationship between the 3 '-VNTR and diallelic apolipoprotein B gene polymorphisms: Haplotype analysis in individuals of European and South Asian origin.</a> <em style='font-style: italic'>Annals of Human Genetics</em>. <b>56</b> (1), 11-33 (1992).</li><li>Kravchenko, S. A., Maliarchuk, O. S., Livshits, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+population+genetics+study+of+the+allelic+polymorphism+in+the+hypervariable+region+of+the+apolipoprotein+B+gene+in+the+population+of+different+regions+of+Ukraine.">A population genetics study of the allelic polymorphism in the hypervariable region of the apolipoprotein B gene in the population of different regions of Ukraine.</a> <em style='font-style: italic'>Tsitologiia i Genetika</em>. <b>30</b> (5), 35-41 (1996).</li></ol>

<p class="jove_content">The authors have no conflict of interest to disclose.</p>

<p class="jove_content">Here we described a simple and cost-efficient method for implementing molecular fingerprinting in undergraduate practical classes.</p>
<p class="jove_content">To screen for genetic variation at the <em>D1S80</em> locus, human biological sample collection along with DNA extraction and analysis is required. It is essential that the ethical use of human biological specimens is ensured throughout the whole process. Sample management is controlled within a comprehensive regulatory framework which ensures the correct use of samples and associated data<sup class="xref">18</sup> (e.g., consent for the use of human biological material). Participants must be properly informed about the use of their samples, the risk of discovery of anomalies in genetic relations (e.g., for related individuals), privacy protection and intentions for future storage of the biological specimens and data. All donors (students or colleagues) must give consent freely and understand the right to withdraw without giving reason. In general, it is indispensable to make oneself familiar with the respective guidelines and regulations for human sample management before conducting this lab class.</p>
<p class="jove_content">For the collection of buccal mucosa epithelial cells in undergraduate laboratory courses, care must be taken to avoid contamination of the DNA samples with DNA from the operator or with DNases. It is recommended that lab coats, gloves and protective glasses are always used as well as sterile swabs and microcentrifuge tubes. Buccal swab-based cell harvesting is considered a convenient and cost-effective method for the collection of genetic material suitable for PCR-based VNTR analysis, as it is relatively inexpensive and noninvasive. Beside buccal swabs, saliva is the most common oral sampling method for medical research. It has been shown that buccal swabs contain a higher proportion of epithelial cells than saliva, making them more reliable in providing sufficient quantity and quality of DNA for PCR-based <em>D1S80</em> VNTR analysis<sup class="xref">19</sup><sup>,</sup><sup class="xref">20</sup>. Additionally, buccal swabs can be mailed out after self-collection overcoming geographical impediments when used, for example, in population diversity studies<sup class="xref">21</sup>.</p>
<p class="jove_content">DNA extraction has become relatively easy due to the development of extraction kits from different companies. Nevertheless, the use of those kits may not be suitable in laboratory classes due to limited financial resources. Here, we presented an easy, fast and cost-efficient method for DNA extraction from human samples without the need of a commercially available DNA extraction kit. The DNA extraction method described does not use organic solvents, instead cellular proteins are removed by increasing the salt concentration using an 8 M potassium acetate solution. This method also allows for handling of several samples at once and yields in sufficient DNA for several genotype analyses for each sample.</p>
<p class="jove_content">PCR is a common technique in many molecular laboratories. While generally trouble-free, there are pitfalls that may complicate the reaction producing spurious results, which have been discussed elsewhere<sup class="xref">22</sup>. PCR conditions presented here have appeared very robust in the face of poor template DNA quality, but lack of PCR amplification was nevertheless occasionally observed when using templates with extremely low DNA concentrations due to poor buccal epithelial cell harvesting. The DNA primers used in this study can be ordered from different companies, such as those cited in the table of materials at the end of this paper. Special care must be taken when working with DNA primers in order to avoid contamination with DNases or DNA from the operator. PCR products should also be kept cold when not in use.</p>
<p class="jove_content">Another critical step is the agarose gel electrophoresis. Fragments differing by only one repeat unit of 16 bp may not be separated in the gel electrophoresis and thus appear as a single band. In this case, a proper determination of the number of repeat units of the <em>D1S80</em> alleles tested cannot be assured. Therefore, the run time of the gel should be increased while the voltage must be reduced and the gel concentration may be increased to provide a better resolution and separation of the single bands.</p>
<p class="jove_content">It is worth mentioning that not all alleles for an VNTR locus contain complete repeat units. Non-consensus alleles (microvariants) that contain incomplete repeat units are common at most VNTR loci and their sizes fall in between sizes of alleles with full repeat units. These microvariants are barely detectable by agarose gel electrophoresis. In contrast, techniques like polyacrylamide gels or capillary electrophoresis can resolve alleles that differ by one to a few repeat units or microvariants<sup class="xref">12</sup><sup>,</sup><sup class="xref">23</sup><sup>,</sup><sup class="xref">24</sup><sup>,</sup><sup class="xref">25</sup><sup>,</sup><sup class="xref">26</sup>. However, the latter techniques are less suitable for undergraduate laboratory classes as they have many disadvantages including use of hazardous compounds, complex preparation and lack of equipment. For the fragment size determination, special care must be taken when measuring the distance of the migrated DNA fragments. If the bands are too diffuse to be measured precisely, the calculation of the <em>D1S80</em> allele fragment size by linear regression may be incorrect resulting in the wrong estimation of repeat unit numbers. In that case a re-run of the agarose gel electrophoresis after optimizing the gel conditions is advisable as previously described by Lorenz and co-workers<sup class="xref">22</sup>.</p>
<p class="jove_content">The entire <em>D1S80</em> VNTR analysis procedure presented here, from buccal epithelial cell harvesting to fragment length evaluation, may be completed in a single working day. This protocol is a robust, cost-efficient and easy-to-use method suitable for undergraduate practical laboratory classes.</p>

<p class="jove_content">Molecular fingerprinting, also referred to as DNA fingerprinting, was introduced by Sir Alec Jeffreys while working in the Department of Genetics at the University of Leicester in 1984<sup class="xref">1</sup>. It is based on the 0.1% of the human genome that differs between individuals and is comprised of approximately three million variants. These unique differences in the genotype allow for the differentiation between individuals and can therefore function as a genetic fingerprint except for monozygotic twins. Consequently, DNA fingerprinting is used for the estimation of relatedness of different individuals that is applied for example in paternity testing or in population diversity studies. In our laboratory classes we aimed to convey the concept of DNA fingerprinting and allele frequencies. The method described here demonstrates a reliable and robust method for genetic fingerprinting by analyzing the Variable Number of Tandem Repeats (VNTR) at the <em>D1S80</em> locus. The method comprises the extraction of DNA from buccal mucosa epithelial cells and subsequent Polymerase Chain Reaction (PCR) to amplify the <em>D1S80</em> locus, followed by visualization of fragment length differences on an agarose gel.</p>
<p class="jove_content">The <em>D1S80</em> VNTR minisatellite locus is highly variable and located at the telomeric region of chromosome 1 (1p36-p35). It was identified and described by Karsai and co-workers in 1990 as a locus with a core repeat unit of 16 bp, allowing for the separation of alleles differing by just one unit<sup class="xref">2</sup>. Furthermore, <em>D1S80</em> shows a high degree of variation with a mutation rate of approximately 7.77 x 10<sup>-5</sup>&#160;<sup class="xref">3</sup>. <em>D1S80</em> has a high degree of heterozygosity<sup class="xref">4</sup><sup>,</sup><sup class="xref">5</sup> (e.g., 80.8% heterozygosity for Caucasians<sup class="xref">6</sup> and up to 87% heterozygosity for African-Americans<sup class="xref">7</sup>). Additionally, the <em>D1S80</em> locus is polymorphic in most populations, with typically over 15 different alleles carrying 14 to 41 repeats each. The frequency of <em>D1S80</em> alleles varies between populations. The allele with 24 repeat units (allele 24) is most frequent in European and Asian populations, whereas allele 21 is most common in African populations<sup class="xref">4</sup><sup>,</sup><sup class="xref">7</sup><sup>,</sup><sup class="xref">8</sup><sup>,</sup><sup class="xref">9</sup><sup>,</sup><sup class="xref">10</sup>. Consequently, the allele frequency distributions are diagnostic for different human populations and need to be taken into account for the estimation of relatedness (e.g., in paternity tests).</p>
<p class="jove_content">PCR based amplification of the<em> D1S80</em> VNTR locus has been a very useful method in forensic science, paternity tests, disease analysis and population diversity studies<sup class="xref">11</sup><sup>,</sup><sup class="xref">12</sup><sup>,</sup><sup class="xref">13</sup><sup>,</sup><sup class="xref">14</sup>. While in forensics today the use of VNTRs has been replaced by short tandem repeats, the <em>D1S80</em> VNTR locus is widely used in the determination of origins and genetic relations among and between populations<sup class="xref">4</sup><sup>,</sup><sup class="xref">8</sup><sup>,</sup><sup class="xref">9</sup><sup>,</sup><sup class="xref">11</sup>. Furthermore, it is often used to teach DNA fingerprinting in practical laboratory classes<sup class="xref">15</sup><sup>,</sup><sup class="xref">16</sup>. The method described here represents a robust, cost-effective and easy-to-use method with a very high success rate in undergraduate laboratory classes. The purpose of this article is to provide an overview of the workflow for the molecular analysis of the human <em>D1S80</em> minisatellite locus from buccal mucosa epithelial cells. It includes demonstration of techniques, simplified protocols and practical suggestions described in previously published works<sup class="xref">2</sup><sup>,</sup><sup class="xref">17</sup>.</p>

<table><tbody><tr><td>1.5 mL microcentrifuge tube</td><td>Sarstedt</td><td>72,706</td><td>autoclaved</td></tr><tr><td>2 mL microcentrifuge tube</td><td>Sarstedt</td><td>7,26,95,500</td><td>autoclaved</td></tr><tr><td>2-propanol</td><td>Fisher chemical</td><td>PI7508/17</td><td></td></tr><tr><td>5x PCR buffer</td><td>Promega</td><td>M7845</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma/Merck</td><td>A6283-500ML</td><td></td></tr><tr><td>Agarose</td><td>BioBudget</td><td>10-35-1020</td><td></td></tr><tr><td>Buccal swabs</td><td>BioBudget</td><td>57-BS-05-600</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td></td><td>5430</td></tr><tr><td>Combs</td><td>BioRad</td><td></td><td></td></tr><tr><td>dNTPs (10 mM)</td><td>Invitrogen</td><td>R0192</td><td></td></tr><tr><td>EDTA</td><td>Carl Roth</td><td>8043.1</td><td></td></tr><tr><td>Ethanol</td><td>Honeywell</td><td>32221-2.5L</td><td></td></tr><tr><td>Gel caster</td><td>BioRad</td><td></td><td></td></tr><tr><td>Gel chamber</td><td>BioRad</td><td></td><td>SubCell GT</td></tr><tr><td>Gel Doc XR+</td><td>BioRad</td><td></td><td></td></tr><tr><td>Geltray</td><td>BioRad</td><td></td><td>SubCell GT</td></tr><tr><td>GeneRuler 50 bp DNA Ladder</td><td>Thermo Fisher</td><td>SM0371</td><td></td></tr><tr><td>Go-Taq Polymerase</td><td>Promega</td><td>M7845</td><td></td></tr><tr><td>Heating block</td><td>Eppendorf</td><td>Thermomixer</td><td>1.5 mL and 2 mL</td></tr><tr><td>Microwave</td><td>Sharp</td><td></td><td>R-941STW</td></tr><tr><td>peqGreen</td><td>VWR</td><td>&amp;nbsp;732-3196&amp;nbsp;</td><td></td></tr><tr><td>Pipettes</td><td>Gilson</td><td></td><td>1000 &amp;micro;L, 200 &amp;micro;L, 20 &amp;micro;L, 2 &amp;micro;L</td></tr><tr><td>Potassium acetate</td><td>Arcos Organics</td><td>217100010</td><td></td></tr><tr><td>PowerPac power supply</td><td>BioRad</td><td></td><td>100-120/220-240V</td></tr><tr><td>Primers</td><td>Sigma/Merck</td><td></td><td></td></tr><tr><td>Scale</td><td>Kern</td><td></td><td>PCB 2.5 kg</td></tr><tr><td>SDS</td><td>Arcos Organics</td><td>218591000</td><td></td></tr><tr><td>Shaker</td><td>IKA labortechnik</td><td></td><td>IKA RH basic</td></tr><tr><td>Tabletop centrifuge</td><td>myFuge</td><td></td><td></td></tr><tr><td>Thermal Cycler</td><td>BioRad</td><td></td><td>C100 Touch</td></tr><tr><td>Tris</td><td>VWR</td><td>103156x</td><td></td></tr><tr><td>Vortex mixer</td><td>LMS</td><td></td><td>VTX-3000L</td></tr></tbody></table>

application of dna fingerprinting using the <em>d1s80</em> locus in lab classes

<p class="jove_content">In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a reliable and robust method for genotyping individuals by Variable Number of Tandem Repeat (VNTR) analysis in the context of undergraduate laboratory classes. The human <em>D1S80</em> VNTR locus is used in this protocol as a highly polymorphic marker based on variation in the number of repetitive sequences.</p>
<p class="jove_content">This simple protocol conveys useful information for teachers and the implementation of DNA fingerprinting in practical laboratory classes. In the presented laboratory exercise, DNA extraction followed by PCR amplification is used to determine genetic variation at the <em>D1S80</em> VNTR locus. Differences in the fragment size of PCR products are visualized by agarose gel electrophoresis. The fragment sizes and repeat numbers are calculated based on a linear regression of the size and migration distance of a DNA size standard.</p>
<p class="jove_content">Following this guide, students should be able to:</p>
<p class="jove_content">&#8226;&#160; Harvest and extract DNA from buccal mucosa epithelial cells<br />
&#8226;&#160; Perform a PCR experiment and understand the function of various reaction components<br />
&#8226;&#160; Analyze the amplicons by agarose gel electrophoresis and interpret the results<br />
&#8226;&#160; Understand the use of VNTRs in DNA fingerprinting and its application in biological sciences</p>

<p class="jove_content">Using the described protocol, <em>D1S80</em> VNTR marker analysis was performed on human genomic DNA extracted from buccal mucosa epithelial cells harvested by swab sampling (<strong class="xfig">Figure 6</strong>). Following the amplification using PCR, a typical representation of an agarose gel containing the <em>D1S80</em> amplicons is shown in <strong class="xfig">Figure 1</strong>. Lane 1 shows the 50 bp molecular weight standard. Next to the molecular weight standard, PCR products from eight student samples are visualized. Lane 10 shows the NTC in which water was used instead of template DNA. Most of the analyzed samples clearly display two bands, representing individuals heterozygous for the <em>D1S80</em> locus. Lane 2 and Lane 9 show a single band representing individuals homozygous for the <em>D1S80</em> locus. Lane 5 is showing an ambiguous single band which is much wider compared to the other bands. This could be a result of two <em>D1S80</em> alleles differing in only one repeat unit. For further analysis, the sample in lane 5 will be considered as a single band, representing a homozygous individual.</p>
<p class="jove_content">Downstream gel electrophoresis fragment analysis performed on verified PCR products was used for size calling of the <em>D1S80</em> VNTR locus of different student samples. Interpretation of the amplicon sizes obtained by PCR analysis relies on the molecular weight standard used. The distance from the well for each band of the molecular weight standard was measured (<strong class="xfig">Figure 1</strong>) and recorded (<strong class="xfig">Figure 2</strong>). Based on the size and migration distance the size of the individual bands can be calculated using a linear regression analysis. The log (base 10) was determined for each band in the student samples (<strong class="xfig">Figure 5</strong>) and the respective antilog represents the number of bp for each amplicon. The number of repeat units can be calculated according to the amplicon size obtained (<strong>Table 2</strong>).</p>
<p class="jove_content">For each student sample the amplicon sizes were calculated by linear regression and each band could be easily assigned to a particular allele as shown in <strong>Table 2</strong>. The information on the <em>D1S80</em> allele sizes can then be used to determine allele frequencies amongst the small population subset of undergraduate students. The 28-repeat allele is the most frequent among the students due to two homozygous individuals for this repeat allele (individuals 1 and 4). The second most frequent allele is the 18-repeat allele, which is carried by the homozygous individual 8 and by the heterozygous individual 6. Low frequency alleles are the 21-, 26- and 30-repeat alleles found only once in individuals 6, 5 and 3, respectively. The allele frequency patterns of the <em>D1S80</em> VNTR locus of the small student population can further be compared to the allele frequency of larger human populations, for example populations originating from different continents<sup class="xref">4</sup><sup>,</sup><sup class="xref">7</sup><sup>,</sup><sup class="xref">8</sup><sup>,</sup><sup class="xref">9</sup><sup>,</sup><sup class="xref">10</sup>.</p>
<p class="jove_content">Among the students, individuals 2 and 3 share the 24-repeat allele, individuals 2 and 7 share the 20-repeat allele and individuals 5 and 7 share the 23-repeat allele. Interestingly, the two <em>D1S80</em> homozygous individuals (individual 1 and 4) share the 28-repeat allele. Matching alleles between two individuals can indicate a potential relatedness. However, shared alleles can also occur by chance. Thus, it is crucial to determine the likelihood of an allele match between two individuals. The likelihood depends on the allele frequency of individual alleles in the general population and statistical approaches should be employed to attach a statistical weight to VNTR marker analysis such as the Bayesian approach. In summary, the presented fingerprinting method can be utilized in hands-on class practicals to teach the use of VNTRs in DNA fingerprinting and its application in biological science.</p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Gel electrophoresis result, DNA separation, lane markers, molecular weight comparison." class="xfigimg" src="/files/ftp_upload/62305/62305fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"><br>
<strong class="xfig">Figure 1</strong><strong>: Representation of an agarose gel after electrophoresis of <em>D1S80</em> amplification products with a ruler placed beside the 50 bp molecular weight standard lane.</strong> Lane 1 contains the 50 bp molecular weight standard, while lanes 2-9 contain PCR reaction products. Lane 10 contains the no template control (NTC). <a href="https://www.jove.com/files/ftp_upload/62305/62305fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Gel electrophoresis data analysis, spreadsheet table with size(bp), distance(cm), log10(bp) calculation." class="xfigimg" src="/files/ftp_upload/62305/62305fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"><br>
<strong class="xfig">Figure 2</strong><strong>: Determination of DNA fragment migration distance and log of each fragment size of the 50 bp molecular weight standard. </strong>The distance from the well (in cm) for each band of the 50 bp molecular weight standard is recorded and the log (base 10) for each fragment is determined. Values are entered into a table using a spreadsheet program. <a href="https://www.jove.com/files/ftp_upload/62305/62305fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Gel electrophoresis graph; log10(bp) vs. Distance(cm); DNA fragment size estimation." class="xfigimg" src="/files/ftp_upload/62305/62305fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"><br>
<strong class="xfig">Figure 3</strong><strong>: Scatter plot generated by plotting DNA fragment migration distance of the molecular weight standard on the x-axis against the log (base 10) of the size of each fragment on the y-axis. </strong><a href="https://www.jove.com/files/ftp_upload/62305/62305fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Linear regression graph, DNA log(bp) vs. distance (cm) with formula y=-0.2255x+3.9013, R²=0.9967." class="xfigimg" src="/files/ftp_upload/62305/62305fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"><br>
<strong class="xfig">Figure 4</strong><strong>: Exploitation of the linear regression line and regression equation as well as the R<sup>2</sup> value for the data.</strong> <a href="https://www.jove.com/files/ftp_upload/62305/62305fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Genetic distance data table; Excel formula calculating allele separation; analysis of homozygous, heterozygous genotypes." class="xfigimg" src="/files/ftp_upload/62305/62305fig05.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"><br>
<strong class="xfig">Figure 5</strong><strong>: Determination of the size of the <em>D1S80</em> amplicons. </strong>The distance from the well for each fragment is entered into the regression equation. The antilog of the y-value represents the fragment size in bp. <a href="https://www.jove.com/files/ftp_upload/62305/62305fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a></p>
<p class="jove_content biglegend" fo:keep-together.within-page="1"><img alt="DNA extraction and PCR analysis diagram showing genomic DNA preparation, VNTR amplification, electrophoresis." class="xfigimg" src="/files/ftp_upload/62305/62305fig6v2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"><br>
<strong class="xfig">Figure 6</strong><strong>: Suggested timeline and workflow for <em>D1S80</em> VNTR analysis. </strong>The entire <em>D1S80</em> VNTR analysis procedure presented here, from buccal epithelial cell harvesting to fragment length evaluation, may be completed within a single working day. <a href="https://www.jove.com/files/ftp_upload/62305/62305fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a></p>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td><strong>Allele size (in bp)</strong></td>
			<td><strong>Number of repeats</strong></td>
		</tr>
		<tr>
			<td>369</td>
			<td>14</td>
		</tr>
		<tr>
			<td>385</td>
			<td>15</td>
		</tr>
		<tr>
			<td>401</td>
			<td>16</td>
		</tr>
		<tr>
			<td>417</td>
			<td>17</td>
		</tr>
		<tr>
			<td>433</td>
			<td>18</td>
		</tr>
		<tr>
			<td>449</td>
			<td>19</td>
		</tr>
		<tr>
			<td>465</td>
			<td>20</td>
		</tr>
		<tr>
			<td>481</td>
			<td>21</td>
		</tr>
		<tr>
			<td>497</td>
			<td>22</td>
		</tr>
		<tr>
			<td>513</td>
			<td>23</td>
		</tr>
		<tr>
			<td>529</td>
			<td>24</td>
		</tr>
		<tr>
			<td>545</td>
			<td>25</td>
		</tr>
		<tr>
			<td>561</td>
			<td>26</td>
		</tr>
		<tr>
			<td>577</td>
			<td>27</td>
		</tr>
		<tr>
			<td>593</td>
			<td>28</td>
		</tr>
		<tr>
			<td>609</td>
			<td>29</td>
		</tr>
		<tr>
			<td>625</td>
			<td>30</td>
		</tr>
		<tr>
			<td>641</td>
			<td>31</td>
		</tr>
		<tr>
			<td>657</td>
			<td>32</td>
		</tr>
		<tr>
			<td>673</td>
			<td>33</td>
		</tr>
		<tr>
			<td>689</td>
			<td>34</td>
		</tr>
		<tr>
			<td>705</td>
			<td>35</td>
		</tr>
		<tr>
			<td>721</td>
			<td>36</td>
		</tr>
		<tr>
			<td>737</td>
			<td>37</td>
		</tr>
		<tr>
			<td>753</td>
			<td>38</td>
		</tr>
		<tr>
			<td>769</td>
			<td>39</td>
		</tr>
		<tr>
			<td>785</td>
			<td>40</td>
		</tr>
		<tr>
			<td>801</td>
			<td>41</td>
		</tr>
	</tbody>
</table>
<p class="jove_content" fo:keep-together.within-page="1"><strong>Table 1: Amplicon size for each </strong><em><strong>D1S80</strong></em><strong> VNTR locus. </strong></p>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td><strong>Individual</strong></td>
			<td><strong>Allele</strong></td>
			<td><strong>Distance (in cm)</strong></td>
			<td><strong>Size (in bp)</strong></td>
			<td><strong>Number of repeat units</strong></td>
		</tr>
		<tr>
			<td>1</td>
			<td>homozygous</td>
			<td>5</td>
			<td>600</td>
			<td>28</td>
		</tr>
		<tr>
			<td>2</td>
			<td>heterozygous</td>
			<td>5.2 ; 5.5</td>
			<td>535, 458</td>
			<td>24, 20</td>
		</tr>
		<tr>
			<td>3</td>
			<td>heterozygous</td>
			<td>4.9 ; 5.2</td>
			<td>626, 535</td>
			<td>30, 24</td>
		</tr>
		<tr>
			<td>4</td>
			<td>homozygous</td>
			<td>5</td>
			<td>600</td>
			<td>28</td>
		</tr>
		<tr>
			<td>5</td>
			<td>heterozygous</td>
			<td>5.1 ; 5.3</td>
			<td>564, 508</td>
			<td>26, 23</td>
		</tr>
		<tr>
			<td>6</td>
			<td>heterozygous</td>
			<td>5.4 ; 5.6</td>
			<td>483, 435</td>
			<td>21, 18</td>
		</tr>
		<tr>
			<td>7</td>
			<td>heterozygous</td>
			<td>5.3 ; 5.5</td>
			<td>508, 458</td>
			<td>23, 20</td>
		</tr>
		<tr>
			<td>8</td>
			<td>homozygous</td>
			<td>5.6</td>
			<td>435</td>
			<td>18</td>
		</tr>
	</tbody>
</table>
<p class="jove_content" fo:keep-together.within-page="1"><strong>Table 2: Allele composition of the different individuals tested. </strong>The number of repeat units can be approximated according to the amplicon size obtained by linear regression analysis.</p>

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Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

<p class="jove_content">Here we describe a simple protocol to generate DNA fingerprinting profiles by amplifying the VNTR locus <em>D1S80</em> from epithelial cell DNA. </p>

Application of DNA Fingerprinting using the <em>D1S80</em> Locus in Lab Classes

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a ...

application-of-dna-fingerprinting-using-the-d1s80-locus-in-lab-classes

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JoVE Journal

Genetics

20.0K Views.  Heinrich-Heine-University. Here we describe a simple protocol to generate DNA fingerprinting profiles by amplifying the VNTR locus D1S80 from epithelial cell DNA. 

Video: Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

Lab Techniques

<h4>Measurement Accuracy</h4>
<p>The ability to repeat an experiment and get the same results, or <em>reproducibility</em>, is essential in scientific research. However, it&#8217;s impossible to repeat an experiment if you don&#8217;t know how you did it. Thus, scientists keep detailed records of their experiments in lab notebooks. These records include important information like the amount of each material used and descriptions of each step of the procedure.</p>
<p>Equipment, like balances and volumetric or graduated glassware, is used to measure solid and liquid compounds for experiments. The precision of each measurement is limited by the equipment used. Balances and other equipment with digital readouts automatically display values at the maximum precision that they can support. The precision of graduated glassware and rulers depends on the increment, which is the amount represented by the smallest distance between two marks.</p>
<p>The surface of most liquids curves upwards at the edges in glass containers. This type of curved liquid surface is called a concave meniscus. When the edges of the liquid curve down, it is called a convex meniscus. When measuring liquid volumes, the reference point for the top of the liquid is the bottom of a concave meniscus or the top of a convex meniscus. To accurately identify the position of the meniscus, liquid volumes are read by looking at the liquid&#8217;s surface from the side at eye-level. Looking at the liquid from above or below will cause the liquid surface level to appear higher or lower than it actually is.</p>
<p>When the reference point of the meniscus touches a mark on graduated or volumetric glassware, the glassware is holding the volume defined for that mark. Volumetric glassware is designed to measure a specific volume, so it only has one mark. Graduated glassware is designed to measure a range of volumes, so it has many marks, or <em>graduations</em>, which are labeled at regular intervals. Both graduated and volumetric glassware may have additional manufacturer-provided information about their precision.</p>
<h4>Calibration and Uncertainty</h4>
<p>Manufacturers calibrate equipment to ensure that the measurements are accurate to within a specified range of uncertainty. For example, a high-precision analytical balance may read out to four decimal places (0.0000 g) with an uncertainty of &#177; 0.0001 g. A reading of 0.0345 g indicates that the true value is between 0.0344 g and 0.0346 g.</p>
<p>Glassware typically has its measurement uncertainty printed on it. To accurately record volume measurements, you must consider both the increment and the uncertainty. For example, a precise volume measurement in a 100 mL graduated cylinder with 1 mL increments and an uncertainty of &#177; 0.5 mL would be recorded to the tenths place (000.0 mL &#177; 0.5 mL). If the reference point of the meniscus falls between two marks, you should estimate a value for the tenths place; otherwise, leave it as 0. Since temperature affects volume, the temperature for which the glassware is calibrated will be printed on it.</p>
<p>Glassware is calibrated either <em>to contain</em> (TC) or <em>to deliver</em> (TD). TC glassware holds the specified volume of liquid when it is filled to the mark, but a small amount of liquid will be left behind when it is poured into another container. Solutions are often prepared in TC glassware because the accuracy of the solution volume affects the accuracy of the concentration. Volumetric flasks are typically calibrated to contain.</p>
<p>TD glassware holds a little more than the amount of liquid specified when it is filled to the mark, but it dispenses only the specified volume. Thus, TD glassware should not be emptied completely when dispensing liquids. This type of glassware is useful for transferring a precise volume of liquid to another container. Both graduated and volumetric pipettes are typically calibrated to deliver.</p>
<p>Unless otherwise specified, the uncertainty of a number is assumed to be on the scale of the last digit. Thus, it&#8217;s particularly important to report the uncertainty of a measurement if the uncertainty is on a larger scale. For rulers and graduated glassware without a known uncertainty, the uncertainty is estimated as half of the smallest increment. The uncertain digit is based on where the meniscus falls between two tick marks.</p>
<h4>Significant Figures</h4>
<p>Significant figures are the numbers in a value that are <em>meaningful</em>, or essential for expressing that value with the appropriate precision. All numbers other than leading zeroes (<strong>0.00</strong>1), trailing zeroes (1,<strong>000</strong>), or scientific notation multipliers (<strong>10</strong><sup>x</sup>) are always significant.</p>
<p>Leading zeroes are never significant because they can be removed by rewriting the number either in scientific notation or, if it is an SI unit, in smaller units. For example, there are only three significant figures in the value 0.00123 m because it can be rewritten as either 1.23 &#215; 10<sup>-3</sup> m or 1.23 mm without losing any information. Note that the scaling factor between different multiples of the same SI unit does <em>not</em> affect the number of significant figures.</p>
<p>Trailing zeroes are significant when they are before or after a decimal point. For example, the measurement 100.110 mL has six significant figures and 100.0 mL has four. Trailing zeroes in a number written without a decimal point are assumed to be insignificant unless they are marked otherwise. There are several different conventions for marking significant zeroes in those cases, but to avoid ambiguity it is advisable to rewrite those values in scientific notation. If the measurement is in SI units, use larger units.</p>
<p>The precision of a measurement limits the precision of any values calculated from it. A calculated value has the same number of significant figures as the <em>least</em> precise measurement or value used in its calculation. If the result has too many significant figures, it is rounded to the appropriate precision. If the result has too few significant figures, it is extended with significant trailing zeroes to preserve the precision. The significant figures of experimentally derived values, like molar mass, density, or some unit conversion factors, must also be considered when they are used in calculations.</p>
<p>The number of significant figures in a calculated value is <em>not</em> limited by physical or mathematical constants in formulas or exact numbers, like the number of data points acquired in an experiment. For example, the formula for calculating the volume of a sphere is V = 4/3&#960;r<sup>3</sup>. The number of significant figures in the calculated volume is <em>only</em> affected by the number of significant figures in the radius measurement <strong>r</strong>. 4/3&#8201; and &#960; are constants, and <sup>3</sup> is simply notation for a mathematical operation.</p>
<p>Defined conversion factors are also treated as constants. For instance, the inch is defined as exactly 25.4 millimeters; therefore, the number of significant figures of the converted value depends only on the original measurement. Always check whether the conversion factor is exactly defined or an experimental value when converting between units.</p>
<p>For simple addition and subtraction of measurements, we use significance arithmetic to determine the number of significant figures. In this case, the answer has as many decimal places as the measurement with the fewest decimal places <em>regardless of the number of significant figures</em>. Thus, 15,643.7 mL + 0.613 mL = 15,644.3 mL.</p>
<p>For multi-step calculations or complex equations that you break into smaller parts to solve, you should keep at least one or two insignificant figures on the intermediate values. For example, if you calculate the values of the numerator and denominator of a fraction separately before dividing them, you should retain the insignificant figures of the numerator and denominator when you divide them. This minimizes <em>rounding error</em>, which is the difference from the number you would get if you entered the entire formula into your calculator at once. If you write down these intermediate values, you should note which digits are insignificant.</p>
<h4>References</h4>
<p>Harris, D.C. (2015). <em>Quantitative Chemical Analysis</em>. New York, NY: W.H. Freeman and Company.</p>

Accuracy, Precision, Uncertainty, and Significant Figures - Concept

Measurement Accuracy
The ability to repeat an experiment and get the same results, or reproducibility, is essential in scientific research. However, ...

Education

JoVE Lab Manual

Chemistry

DNA Isolation and Restriction Enzyme Analysis

<p>The revelation of DNA as the hereditary molecule in all organisms has led to enormous scientific and medical breakthroughs and significantly enhanced our understanding of ourselves and other organisms. DNA isolation and profiling have been the fundamental first steps for many of the advancements in the past century; from identification of gene function, to revolutions of agriculture and forensics.</p>

<h4>Isolating DNA from Cells</h4>

<p>DNA isolation is a fairly simple procedure that requires few but essential steps: cell harvesting, lysis, protein degradation, and finally DNA precipitation. Generally, DNA can be harvested from small amounts of tissue, such as a single leaf of a plant, hair follicles of animals, or epithelial cells that can be easily sloughed off within the mouth with a simple saline solution. Since DNA is compartmentalized inside the nucleus or mitochondria within eukaryotic cells, scientists first break apart the cell and nuclear membrane to expose the DNA, which is done with the lysis step. However, once DNA is exposed, it can be degraded by heavy metal ions and extremes of pH. Therefore, a detergent-containing buffer solution is used to not only dissolve the cell membrane, but also to protect DNA from degrading elements in the solution. Moreover, DNA is an unusually long molecule that is condensed inside the nucleus by packing tightly around special proteins called histones. Therefore, an enzyme called Proteinase K, which degrades peptide bonds of proteins, is added to separate the DNA from histones and other proteins. Next, scientists separate DNA from the rest of the cellular extract by using its chemical properties. Due to the negatively charged structure of nucleic acids, Na<sup>+</sup> ions in a sodium chloride solution bind to and integrate into entangled DNA strands. Once the DNA is clumped, it can be observed by the naked eye. DNA is insoluble in alcohol and readily precipitates at low temperatures, thus scientists add cold alcohol to precipitate DNA. Finally, the precipitated DNA can be dissolved and stored in deionized water or a mild buffer, free of any enzymes that can degrade nucleic acids, until it is used.</p>

<h4>Quantifying DNA</h4>

<p>Reactions that use DNA often require high purity and precise amounts of DNA. Therefore, following DNA isolation, scientists use a spectrophotometer to quantify the amount of DNA in their sample, as well as the purity of the DNA in the sample. A spectrophotometer measures the intensity of a light beam passed through a sample as a function of its wavelength. Nucleic acids absorb light at 260 nm, whereas proteins absorb light at 280 nm. By measuring the intensity of a light beam at 260 nm, scientists can determine the DNA concentration. Furthermore, by assessing the light intensity at both 260 nm and 280 nm, the purity of the DNA content can be determined. In a sample of pure DNA, the 260/280 nm ratio should approach the value of 2.</p>

<h4>Restriction Enzymes</h4>

<p>For a number of analyses, such as cloning, sequencing, and mapping DNA fragments and genomes, the isolated DNA needs to be cut at specific sequences using restriction enzymes. These enzymes are produced by bacteria as mechanisms of innate immunity against intruding viral DNA and function as molecular scissors. Hence, after restriction enzymes identify a viral DNA, they start to break it apart by cutting it into segments at certain nucleotide sequences they recognize. These nucleotide sequences are often six to twelve nucleotides long and are generally palindromic, which means that they have the same nucleotide sequence in the 3&#8217;-5&#8217; and 5&#8217;-3&#8217; directions. For instance, the restriction enzyme EcoRI cuts DNA at the sequence 3&#8217;...GAATTC&#8230;-5&#8217;, which reads the same in the 5&#8217;-3&#8217; direction. Like EcoRI, there are hundreds of enzymes that correspond to many distinct palindromic sequences. Once DNA is cut by these enzymes, scientists can load the DNA onto a gel in an electrophoresis chamber and pass electrical current through the gel. DNA carries negative charges, which causes DNA fragments to migrate towards the anode. However, the pores of the gel slow down larger DNA fragments relative to smaller DNA sequences, thus the pieces separate according to their size. After the completion of the electrophoresis, DNA fragments can be visualized with the aid of dyes that specifically bind to DNA molecules.</p>

<p>Cutting DNA with restriction enzymes and separating DNA fragments with electrophoresis allows researchers to identify the sizes of unknown fragments relative to a standard ladder. These fragments of interest can then be further isolated and ligated into a plasmid vector for genetic cloning. Genetic engineering and laboratory analyses have been improved immensely with these methods. With DNA profiling, scientists are able to sequence genomes to further understand the workings of organisms and are also able to identify and characterize certain genes<sup>1</sup>.</p>

<p>DNA isolation and profiling are also practical for events directly impacting everyday life. For example, forensics scientists routinely analyze DNA samples to provide evidence for criminal and civil cases<sup>2</sup>. Similarly, healthcare workers extract and analyze DNA to check for paternity and genetic diseases. Recently, DNA ancestry analysis kits became popular by allowing users determine their genetic lineage, find relatives, and even discover their genetic disposition to various health conditions<sup>3</sup>. Finally, with advancements in biotechnology and genetic engineering, personalized medicine is gaining traction among clinical researchers to develop treatments by using an individual&#8217;s genetic data, paving the way to effectively treat patients without detrimental side effects<sup>4</sup>.</p>

<h4>References</h4>

<ol class="references">
	<li><strong>Chambers, GK, et al.</strong> DNA fingerprinting in zoology: past, present, future. <em>Investig Genet. </em>2014, Vol. 5, 3 doi: 10.1186/2041-2223-5-3.</li>
	<li><strong>Roewer, L.</strong> DNA fingerprinting in forensics: past, present, future. <em>Investig Genet. . </em>2013, Vol. 4, 22 doi: 10.1186/2041-2223-4-22.</li>
	<li><strong>Kirkpatrick, BE and Rashkin, MD.</strong> Ancestry Testing and the Practice of Genetic Counseling. <em>J Gen Counseling. </em>2016, Vol. 26, 1 (6-20).</li>
	<li><strong>Hamburg, MA and Collins, FS.</strong> The Path to Personalized Medicine. <em>N Engl J Med . </em>2010; , Vol. 363, 301-304.</li>
</ol>


The revelation of DNA as the hereditary molecule in all organisms has led to enormous scientific and medical breakthroughs and significantly enhanced our ...

Biology

Evolutionary Relationships

<p>Humans have been attempting to properly classify living things since Aristotle made the first attempt during the 4<sup>th</sup> century BC. Aristotle&#8217;s system was improved upon during the Renaissance and then, subsequently, by Carolus Linnaeus in the mid 1700&#8217;s. These more formal classification and organization systems grouped species by their physical similarity to one another. For example, all vertebrates have a backbone, but invertebrates do not. Traits like the backbone are called synapomorphies, which are traits that are shared by a group of organisms, presumably because they were derived from a common ancestor. As we will explore, this method has been shown to have limitations and has more recently been amended to include genetic analysis. Still, scientists construct trees called dendrograms to create a visual representation of how species are related to one another and share common ancestors. These dendrograms can aid in our understanding of the evolutionary processes that drive these relationships. Genetic comparisons have added an important tool guiding the analysis of evolutionary relationships.</p>
<h4>Types of Dendrogram</h4>
<p>A type of dendrogram, called a cladogram, depicts the hypothetical genealogical relationships between species with the tips (or leaves) of the tree representing a species and the branches showing how species are related to each other. A slightly more complicated type of tree, called a phylogram, differs from a cladogram in that the branches leading to the species are of different lengths. The length of a branch in this type of tree represents the degree of change between species: the longer the branch, the more time since the species have diverged from a common ancestor. In both types of tree, the common ancestor of a group of species is indicated by a node, which is the point where a series of branches meet. Species that are more closely related to each other (most recently shared a common ancestor) will be located closest to the node. The two species that share a node are called a sister group<sup>1</sup>.</p>
<h4>Understanding Evolutionary Relationships using Genetic Data</h4>
<p>Historically, cladograms were constructed by comparing the morphology (physical structure) of organisms. This method is still practiced but the techniques have been modernized to include comparison of DNA (deoxyribonucleic acid) sequences between species. Using DNA for building trees has several advantages over relying solely on morphology, including being able to calculate an estimate of how long ago different species shared a common ancestor<sup>1</sup>. However, using DNA is not always feasible, especially when trees include extinct organisms. DNA is best found in soft tissues, which are not preserved during the fossilization process, and therefore it is uncommon for a DNA sample of an extinct species to be available.</p>
<p>DNA is passed on from parents to their offspring in hereditary units called genes. The nucleotide (A, G, C, and T) sequence of genes found in different species are frequently quite similar, likely due to their having come from a common ancestor. This fact allows researchers to align sequences from different species with one another to build the trees described above. Species with more similarity between their nucleotide sequences will be placed next to each other in a tree, and species with less sequence similarity will be placed further apart from each other.</p>
<p>Bioinformatics are the tools used by biologists to analyze large datasets using a combination of computer science, mathematical modeling, and statistics. One such tool is called BLAST (Basic Local Alignment Search Tool), which can be used to quickly search the entire genome of any species that is available in the NCBI (National Center for Biotechnology Information) database<sup>2</sup>. The NCBI database combines several different databases that hold different types of DNA sequence information. The process of a BLAST search includes complex computer algorithms, but basically, BLAST aligns sequences of each nucleotide base from a submitted DNA sequence (known as the <em>query</em> sequence) with sequences in the data base that most closely match it. The DNA sequences that are found will be listed in order of similarity to the sequence in question, and will therefore be from species closely related to the species containing the query gene. This comparison may or may not depict the actual evolutionary relationship between species because genes evolve at different rates. Additionally, genomes sometimes contain more than one instance of a similar sequence.</p>
<p>Comparison of DNA sequences of genes is valuable beyond consideration of evolutionary relationships. Frequently, genes are identified in model organisms, such as the fruit fly, <em>Drosophila melanogaster</em>, or the mouse<sup>3</sup>. Integral to studying a gene, the function of its product is commonly identified and analyzed. If a researcher is interested in studying that function in a different organism (humans for example), BLAST or other bioinformatic tools can be used to find candidate genes based on their similarities to the genes of known function from model organisms.</p>
<p>Human genes can also be used as the starting point to find homologs in model organisms. In fact, human disease research depends heavily on this. Once a human gene of interest is identified, mice can be genetically manipulated to have the homologous gene disrupted, or &#8220;knocked out,&#8221; creating a model of the human disease that can be studied in order to understand and treat the disease. There are many of these mouse strains currently available. For example, there is a mouse model for human Cystic Fibrosis (CF) called the <em>Cftr</em> knockout mouse and another modeling atherosclerosis, called the <em>Apoe</em> knockout<sup>3</sup>.<br />
 </p>
<h4>References:</h4>
<ol class="references">
	<li><strong>Annenberg Learner.</strong> Online Textbook: Unit 3 Evolution and Phylogenetics. <em>Rediscovering Biology. </em>[Online] 2017. [Cited: August 23, 2018.] https://www.learner.org/courses/biology/textbook/compev/compev_3.html.</li>
	<li><strong>National Center for Biotechnology Information, U.S. National Library of Medicine.</strong> BLAST: Basic Local Alignment Search Tool. [Online] https://blast.ncbi.nlm.nih.gov/Blast.cgi.</li>
	<li><strong>National Human Genome Research Institute.</strong> Background on Mouse as a Model Organism. <em>National Human Genome Research Institute. </em>[Online] December 2002. [Cited: August 23, 2018.] <a href="https://www.genome.gov/10005834/background-on-mouse-as-a-model-organism/" target="__blank">https://www.genome.gov/10005834/background-on-mouse-as-a-model-organism/</a>.</li>
</ol>

Evolutionary Relationships: Using BLAST to Test Evolutionary Hypotheses - Concept

Humans have been attempting to properly classify living things since Aristotle made the first attempt during the 4th century BC. Aristotle&#8217;s system ...

Evolution

Identifying Alcohols

<p>Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of Reagents<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Before you begin, put on a lab coat, safety glasses, and nitrile gloves, and set up a waste container for aqueous chromium, iron, and zinc waste. <strong>Note:</strong> Work in a fume hood because the reagents are acidic, toxic, and corrosive. The reagents undergo exothermic reactions when they are mixed, so you will prepare them in ice baths.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare 100 mL of 0.03 M aqueous iron(III) chloride. First, weigh out 0.5 g of anhydrous iron(III) chloride and pour it into a 250-mL beaker.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Place about 250 mL of crushed ice in a 600-mL beaker and add just enough water to cover the ice. Settle the beaker of iron(III) chloride in the ice bath and place the bath on a stir plate.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Measure 100 mL of deionized water and pour it into the beaker containing iron(III) chloride. Add a medium stir bar to the beaker and stir until the iron(III) chloride has completely dissolved.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Label a brown 100-mL glass bottle &#8216;0.03 M iron(III) chloride in water&#8217;. Once the iron(III) chloride has dissolved, retrieve the stir bar, and pour the solution into the labeled bottle. Cap the bottle and store it in a cool, dry, dark cabinet for acids.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Prepare 100 mL of Jones reagent. Measure out 75 mL of deionized water, pour it into a 250-mL beaker, and add a medium stir bar. Refresh the ice bath and chill the water in the bath.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Measure out 25 g of chromium trioxide in a fume hood and place it in a clean 150-mL beaker.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Make a second ice bath and settle the beaker of chromium trioxide in it. Put this bath on the stir plate and add a medium stir bar to the beaker.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Measure out 25 mL of 98% by weight sulfuric acid and start slowly adding it to the chromium trioxide. When enough sulfuric acid has been added to cover the stir bar, start slowly stirring the mixture, being careful to avoid splashing.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Once you have added all 25 mL of sulfuric acid, continue stirring the mixture until the chromium trioxide has dissolved completely. Then, turn off the stir motor, and remove the bath and mixture from the stir plate.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Now, confirm that the water in the ice bath is cold. Place that bath on the stir plate and start stirring the water at a moderate speed.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Use a glass Pasteur pipette to slowly add the chromium-sulfuric acid mixture to the cold water. Leave the Pasteur pipette in the empty beaker when you are done.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Continue stirring the mixture until the solution cools to room temperature. Then, turn off the stir motor and remove the bath from the stir plate.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Transfer the stir bar to the empty beaker without letting any liquid drip on the floor of the hood. Carefully pour the Jones reagent into a 100-mL brown glass bottle and cap it. Label the bottle and list its components. Then, store it in the acid cabinet.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Jones reagent contains hexavalent chromium, which is highly toxic and corrosive, so you must quench the residual solution before you clean the glassware. With the beaker in the ice bath, slowly add 10 &#8211; 20 mL of water.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Pipette water along the sides of the beakers to rinse down the residues. Then, slowly add cyclohexanol to the beaker until the solution lightens in color. Quench the residue on other glassware and tools the same way.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Lastly, prepare about 50 mL of Lucas reagent. Measure 47 mL of 37% by weight hydrochloric acid and pour it into a 100-mL beaker.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Prepare a fresh ice bath and set it on the stir plate. Place the beaker in the bath, add a stir bar, and start stirring the HCl.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">While the HCl cools, weigh out 76.9 g of anhydrous zinc chloride. Use a spatula to slowly add the zinc chloride to the cold HCl, pausing as needed to let the mixture cool back down.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Continue stirring until the zinc chloride has dissolved completely and the solution has cooled to room temperature.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">While you wait, label a 100-mL glass bottle as &#8216;Lucas reagent&#8217;. When the solution is ready, turn off the stir motor, retrieve the stir bar, and pour the solution into the labeled bottle. Close the bottle with an acid-resistant cap and store it in the acid cabinet.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Preparation of the Laboratory<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Place bottles of 1-butanol, 2-butanol, and 2-methyl-2-propanol in a hood for solvents.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Put a box of Pasteur pipettes and a few pipette bulbs in the same hood and set a glass waste container within easy reach.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Then, pour about 20 mL of acetone in a 50-mL glass bottle. Cap the bottle, label it, and put it in the solvent hood.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Next, set a container of phenol by an analytical balance. Confirm that there are enough small spatulas, weighing boats, and laboratory wipes for the lab.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Now, randomly assign each lab group one of the four unknown alcohols: 1-propanol, 2-propanol, 2-methyl-2-butanol, and 4-chlorophenol.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">For each group, prepare a vial of about 1 mL, or 0.5 g, of the appropriate alcohol and label it with a code name. Store the vials in the instructor&#39;s hood.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Set out the following glassware and equipment at each student lab station (we suggest that students work in pairs):
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;13&#160;&#160;&#160;&#160;Small test tubes</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Test tube rack</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;3&#160;&#160;&#160;&#160;Small vials with caps</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;50-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;250-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;5-mL graduated cylinder</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Glass stirring rod</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Roll of labels or lab tape</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Labeling pen/marker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;Pipette bulbs</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Fill 250-mL plastic wash bottles with deionized water and place one at each workstation. Set out a box of Pasteur pipettes in a central area for students to use at their hoods.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Shortly before the lab, place the three test reagents in a central fume hood. Give the unknowns to the student groups when they are ready to start the lab.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">After the lab, collect the orange solutions containing unreduced chromium(VI) from the Jones test and quench them with water and isopropyl alcohol.</div>
		</li>
	</ul>
	</li>
</ol>

Identifying Alcohols: Ferric Chloride Test, Jones Test, and Lucas Test - Prep

Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA

	Preparation of ReagentsExpand
	Here, we show the laboratory preparation for ...

Lab Manual Sub Articles

Identification of Unknown Aldehydes and Ketones

<p>Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">DNPH Test for Aromatic Aldehydes and Ketones<button class="expand">Expand</button>
	<p>In this lab, you&#39;ll use the DNPH test, the Tollens&#39; test, and the iodoform test to identify two unknown aldehydes or ketones. You&#39;ll use butanone and benzaldehyde as known compounds to confirm that the tests are working as expected.</p>
	<p>You&#39;ll start the lab with the DNPH test, which relies on the reaction between 2,4-dinitrophenylhydrazine, or DNPH, and aldehydes and ketones. When you combine DNPH and an aldehyde or ketone in an acidic environment, they undergo a condensation reaction to form the corresponding hydrazone. The hydrazone then precipitates from solution. If the precipitate is red to orange, the aldehyde or ketone was aromatic. If it&#39;s orange to yellow, the aldehyde or ketone was non-aromatic or aliphatic.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Put on a lab coat, safety glasses, and nitrile gloves.<strong> Note: </strong>Many chemicals in this lab are toxic, corrosive, or flammable, so you will perform this lab in a fume hood. Change your gloves if you get any chemicals on them.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Get a vial of each known compound and choose two unknown compounds to analyze. Write the codes for your unknowns in your lab notebook.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Label a 250-mL beaker for aqueous waste and a 600-mL beaker for used pipettes. Put several clean Pasteur pipettes in another beaker and get more whenever you need them.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Obtain four small test tubes in a rack and label them with the four compound names, or codes, and the name of the first test.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Bring a 50-mL beaker and a graduated cylinder to the reagent hood and obtain 8 mL of the DNPH solution.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Use four Pasteur pipettes to place 3 &#8211; 4 drops of each compound into its test tube. <strong>Note:</strong> Don&#39;t reuse pipettes during this lab because cross-contamination will invalidate the test results.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Add 2 mL of the DNPH solution to one of the test tubes and mix it well with a glass rod. Thoroughly rinse the glass rod with deionized water over your aqueous waste beaker to remove all traces of the ketone or aldehyde.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Add 2 mL of the DNPH solution to each of the other three test tubes in the same way. Make sure to thoroughly clean the glass rod after each one. When you&#39;re finished, let the solution sit for 15 min.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">While you wait, set up for vacuum filtration and fill a 250-mL beaker with deionized water.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">After 15 minutes, inspect the precipitates in the test tubes. <strong>Note:</strong> Benzaldehyde has an aromatic group, so you should see a red to orange solid in its test tube. Butanone is aliphatic, so you should see an orange to yellow solid in its test tube. Record the results in your lab notebook.
		<h4><strong>Table 1: DNPH, Tollens&#39;, and Iodoform Results</strong></h4>
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Compound</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>DNPH </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Tollens&#39; </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Iodoform </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Solubility in water </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Hydrazone m.p. </strong></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">Butanone</td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">Benzaldehyde</td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160; &#160;</td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160; &#160;</td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/69/Table_1._DNPH,_Tollens_,_and_Iodoform_results.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Compare the solids from the unknown compounds to the known compounds. For each unknown, record whether this solid was more red-orange, like benzaldehyde, or more orange-yellow, like butanone.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Use a clean Pasteur pipette to wet the filter paper of the vacuum filtration setup with deionized water and collect the solid from one of your unknowns by vacuum filtration. Rinse out any solid left in the tube with deionized water.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Wash the solid on the filter paper with 10 mL of deionized water.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Pick up a tiny amount of the solid with a clean glass rod and smear it on pH paper. If the pH is below 6.5, rinse the solid with another 10 mL of deionized water. Keep rinsing the solid and checking its pH until it is between 6.5 and 7.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Label a small watch glass for the unknown and carefully transfer the filter paper and solid to the watch glass to dry.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Thoroughly rinse the B&#252;chner funnel with deionized water, dispose of the filtrate in the DNPH solution waste, and reassemble the vacuum filtration setup with a new piece of filter paper.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Collect and wash the solids from the second unknown compound and from the two known compounds in the same way.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Leave the four solids in your fume hood to dry (1 h).</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">The Tollens&#8217; Test<button class="expand">Expand</button>
	<p>When you combine an aldehyde with diammine silver(I), or Tollens&#39; reagent, the reagent is reduced to metallic silver and ammonia. The silver either coats the inside of the reaction vessel or precipitates as a black solid, so it&#39;s easy to see that the reaction happened.</p>
	<p>Most ketones are harder to oxidize than aldehydes and therefore don&#39;t react with the Tollens&#39; reagent. For this lab, you can assume that if silver appears, the compound is an aldehyde. If silver does not appear, the compound is a ketone.</p>
	<p>The Tollens&#39; reagent solution contains a strong base and must be disposed of specially to avoid forming an explosive decomposition product. Handle it carefully and remember to put the Tollens&#39; test waste in the designated container.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-4">
		<div class="lab-step">Place your used test tubes in a beaker and label four new tubes with the compound names or codes and Tollens&#39; test.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Bring a clean 50-mL beaker and a 5-mL graduated cylinder to the reagent hood and obtain 4 mL of Tollens&#39; reagent.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Use clean pipettes to place 3 &#8211; 4 drops of each compound in its test tube.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Add 1 mL of Tollens&#39; reagent to each test tube and mix the solutions well with a clean glass rod, thoroughly rinsing the rod with deionized water between tubes. Set the rack of tubes to the side and let it sit for 10 min.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">While you wait, get about 100 mL of water in a 150-mL beaker and set it on a hotplate. Position a small 3-prong clamp above the beaker.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Check the benzaldehyde tube after 10 min.<strong> Note:</strong> Benzaldehyde should have a positive result, as indicated by a silver film or precipitate. If you don&#39;t see a positive result in the benzaldehyde tube, secure a thermometer to the test tube and heat in the water bath at 35 &#176;C for 5 min. The silver film, or precipitate, should then appear. Record this result in your lab notebook.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Butanone won&#39;t react with Tollens&#39; reagent. <strong>Note: </strong>The butanone tube is an example of a negative Tollens&#39; test result. Record this in your lab notebook.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Check the tubes of your unknowns. If you see a silver mirror or precipitate, record a positive Tollens&#39; test result for that unknown. If you don&#39;t, heat the tube in the water bath for 5 min and check again. If the test is still negative, record that result in your lab notebook.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">When you&#39;re done, turn off the hotplate and dispose of the Tollens&#39; test solutions in the appropriate waste container. Put silver-coated tubes in the collection box for acid cleaning and put the rest in your beaker.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">Iodoform Test for Methyl Ketones<button class="expand">Expand</button>
	<p>When you combine iodine and a methyl ketone in aqueous base, iodine replaces the alpha methyl&#39;s hydrogens. An addition-elimination reaction followed by proton transfer yields iodoform and a carboxylate anion. Iodoform is insoluble in water, so you&#39;ll see a yellow precipitate if the compound that you&#39;re testing is a methyl ketone. If it is not a methyl ketone, the test solution will eventually turn brown because of the unreacted iodine.</p>
	<ul class="lab-items">
		<li data-sub-note-item="3-2">
		<div class="lab-step">Before the iodoform test, check the solubility of all four compounds in water. Label four clean test tubes with the compound names or codes and H<sub>2</sub>O. Label another four test tubes with the compound names and CHI<sub>3</sub>.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Use clean Pasteur pipettes to place 3 &#8211; 4 drops of each compound in its test tube.</div>
		</li>
		<li data-sub-note-item="3-4">
		<div class="lab-step">Add 2 mL of deionized water to each labeled tube. Mix well with a clean glass rod. Thoroughly clean the rod with deionized water between tubes.</div>
		</li>
		<li data-sub-note-item="3-5">
		<div class="lab-step">Wait a few minutes for the liquids to settle before evaluating them. <strong>Note:</strong> Butanone is moderately soluble in water, so you should see little to no separation. Benzaldehyde is sparingly soluble in water, and its density is a little higher than water&#39;s. You should see a small layer at the bottom of the tube.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Inspect your unknowns for liquid layers and record the results for all four compounds in your lab notebook.</div>
		</li>
		<li data-sub-note-item="3-7">
		<div class="lab-step">Add 2 mL of deionized water to the butanone tube and any water-soluble ketones and mix them well with a clean glass rod. <strong>Note: </strong>If you have an unknown ketone that is insoluble in water, instead add 2 mL of 1,2-dimethoxyethane and mix it well with a clean glass rod.</div>
		</li>
		<li data-sub-note-item="3-8">
		<div class="lab-step">Now, perform the iodoform test, which is for ketones only. Use a clean 50-mL beaker and a small graduated cylinder to obtain 4 or 6 mL of 3 M NaOH, depending on whether you have two or three ketones to test.</div>
		</li>
		<li data-sub-note-item="3-9">
		<div class="lab-step">Pour ~10 mL of iodoform reagent into another 50-mL beaker.</div>
		</li>
		<li data-sub-note-item="3-10">
		<div class="lab-step">Add 2 mL of 3 M NaOH to each ketone tube and mix well, thoroughly cleaning the glass rod with deionized water between each tube.</div>
		</li>
		<li data-sub-note-item="3-11">
		<div class="lab-step">Obtain a clean Pasteur pipette and add the iodoform reagent dropwise to the butanone tube, frequently stirring until you see a yellow precipitate. <strong>Note:</strong> Butanone is a methyl ketone, so it will produce a positive result.</div>
		</li>
		<li data-sub-note-item="3-12">
		<div class="lab-step">Rinse the glass rod with deionized water and start adding the iodoform reagent dropwise to your first, or only, unknown ketone, while stirring frequently. Continue adding the reagent until either yellow iodoform precipitates, like you saw with butanone, or the solution stays brown, even after you stir it well. <strong>Note: </strong>The latter is a negative result.</div>
		</li>
		<li data-sub-note-item="3-13">
		<div class="lab-step">If you have another unknown ketone, clean the glass rod well and repeat the test with your second ketone.</div>
		</li>
		<li data-sub-note-item="3-14">
		<div class="lab-step">When you&#39;re finished, dispose of your iodine-containing solutions in the appropriate waste container.</div>
		</li>
		<li data-sub-note-item="3-15">
		<div class="lab-step">Once the solids from the DNPH test are dry, measure the melting point of each solid and record them in your lab notebook. Put the remaining solutes in the waste container for solid DNPH derivatives.</div>
		</li>
		<li data-sub-note-item="3-16">
		<div class="lab-step">Dispose of the leftover organic liquids and the aqueous aldehyde mixtures in the usual waste containers.</div>
		</li>
		<li data-sub-note-item="3-17">
		<div class="lab-step">Rinse the Pasteur pipettes used for the aldehydes and ketones with acetone before putting them in the glass waste. Pour your aqueous waste down the drain with running tap water. Clean your glassware, put away your lab equipment, and collect and throw out any trash in your fume hood before you leave.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="4" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="4-1">
		<div class="lab-step">Identify your unknown compounds. There are six options: acetophenone, butanal, 3-methyl-2-butanone, pentanal, 2-pentanone, and 3-pentanone.</div>
		</li>
		<li data-sub-note-item="4-2">
		<div class="lab-step">Look at the DNPH results. If the precipitate was red to orange, the unknown aldehyde or ketone is aromatic. If it was yellow, the unknown is aliphatic. <strong>Note:</strong> Since only one possible compound is aromatic, red precipitate means that the unknown was acetophenone. As confirmation, you should also have seen a negative Tollens&#39; test and a positive iodoform test.</div>
		</li>
		<li data-sub-note-item="4-3">
		<div class="lab-step">A yellow precipitate in the DNPH test means that the unknown could be any of the other five compounds.</div>
		</li>
		<li data-sub-note-item="4-4">
		<div class="lab-step">Look at the results of the Tollens&#39; test. If you saw a silver mirror or silver-gray precipitate in the test tube, the unknown compound is an aldehyde. If you didn&#39;t see any change, then the unknown compound is a ketone. If your unknown is an aldehyde, it could be pentanal or butanal.</div>
		</li>
		<li data-sub-note-item="4-5">
		<div class="lab-step">Compare the melting point of the unknown&#39;s hydrazone derivative to literature values for each aldehyde to figure out which one it was.</div>
		</li>
		<li data-sub-note-item="4-6">
		<div class="lab-step">If Tollens&#39; test was negative and the DNPH test showed an aliphatic compound, the unknown could be 3-methyl-2-butanone, 2-pentanone, or 3-pentanone.</div>
		</li>
		<li data-sub-note-item="4-7">
		<div class="lab-step">Check the results of the iodoform test to narrow it down. If you saw solid yellow iodoform, the compound was a methyl ketone. If you saw a brown solution, it was not a methyl ketone.</div>
		</li>
		<li data-sub-note-item="4-8">
		<div class="lab-step">Only one of the aliphatic ketones is not a methyl ketone, so if you saw a brown solution, the unknown was 3-pentanone. If you saw yellow iodoform precipitate from solution and the DNPH test also had yellow precipitate, then the unknown is 3-methyl-2-butanone or 2-pentanone.</div>
		</li>
		<li data-sub-note-item="4-9">
		<div class="lab-step">Use the melting point of the unknown&#39;s hydrazone derivative to figure out which methyl ketone you had.</div>
		</li>
	</ul>
	</li>
</ol>

Test for Aldehydes and Ketones: DNPH, Tollen's Test, and Iodoform Test - Procedure

Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

	DNPH Test for Aromatic Aldehydes and KetonesExpand
	In ...

Genetics of Organisms

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">REF and WEF Fly Genetics
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: At your station, you should have four vials of flies. They will be labeled as male or female. They will also be labeled with the notation REF for red-eyed flies or WEF for white-eyed flies. The female vial should also be labeled virgin as these flies should be unmated for the purpose of this experiment.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Make sure you have around five flies in each vial. NOTE: You will perform crosses using these flies to determine if red or white eyes is dominant and if the trait is sex-linked. You should also have two fly anesthesia wands, a paint brush, magnifying glass, fly anesthetic, some empty fly vials, and a sheet of white paper.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Draw a line in the middle of the paper to make a male side and a female side.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Dip the fly anesthesia wand into the solution and insert it into the red-eyed female vial until the flies stop moving.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Then, dip the same wand into the white-eyed male vial until these flies are also anesthetized.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Tap the flies from the two vials onto the sheet - Females on one side and males on the other.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Check to make sure that all the flies from the female vial are female and likewise, confirm that the male flies are male.  NOTE: Males are slightly smaller and have a darker solid pigment on the end of their abdomen. Females are larger and have stripes rather than a solid spot.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">If there are any male flies in the female vial, obtain a new vial as these females likely have sperm from males from their own strain and are unusable.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Now, move the five red-eyed females and five white-eyed males into a new vial using the paintbrush.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Label this vial to indicate that this is a cross between red-eyed females and white-eyed males. HYPOTHESES: Use Punnett squares to formulate hypotheses for this experiment. Consider what you will see in a few days if the white allele is dominant and there is no effect of parental sex. What if red is dominant? Under the potential offspring genotype in each box, write the expected phenotype.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">To set up the reverse cross, again dip the fly anesthesia wand into the solution and insert it this time into the white-eyed female vial and then the red-eyed male vial until all the flies have stopped moving.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Tap the flies onto a sheet of white paper and check to make sure that all the flies from the female vial are female and likewise with the male flies.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Now, move the five white-eyed females and five red-eyed males into a new vial using the paintbrush.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Label this vial to indicate that this is a cross between white-eyed females and red-eyed males.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Finally, place the two tubes containing the new crosses into a rack for incubation at room temperature. NOTE: After about three days, larvae should be moving around in the vial.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Remove the 10 adult flies and dispose of them into the morgue tube containing 70% ethanol.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Now, put the tubes back in the rack and let the larvae mature until they reach the adult stage in up to two weeks.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Once all of the larvae from these crosses have matured to adulthood, anesthetize the flies in one of the tubes using the wand.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Then, on a piece of white paper, sort the flies by sex and phenotype and then record these data.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Now, repeat this process and tally the sexes and phenotypes for the second cross (steps 1 &#8211; 18).
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Finally, once you have counted all of your flies, dispose of them in the morgue vial.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To begin investigating your results, first compare the data you collected with the hypothesis you made using the Punnett squares.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Did your data match the simple Punnett square hypothesis where red or white is dominant and there is no effect of sex? Compare your data with other lab groups.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">If your data does not match what your Punnett squares predicted, does this indicate that the gene may be sex-linked?
</div></li><li data-sub-note-item="2-4"><div class="lab-step">If the red gene were dominant and sex-linked, when we cross red-eyed females with white-eyed males, all of the offspring would have red eyes. Assuming all of the females were homozygous for the color allele, does this match your data for the first cross?
</div></li><li data-sub-note-item="2-5"><div class="lab-step">If white were dominant and the gene were sex-linked, we would see the exact opposite. So all of the flies would have white eyes. From your data, can you conclude from your first cross whether red or white is dominant?
</div></li><li data-sub-note-item="2-6"><div class="lab-step">When the reciprocal cross of white-eyed females and red-eyed males is performed, if the red phenotype is dominant, all of the females would be red and all of the males would be white. This would also confirm that the gene is specifically x-linked because white-eyed females could then only be produced in crosses with white-eyed males. Did your second cross produce any white-eyed females?
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Finally, if the gene were x-linked and white was dominant, crossing white-eyed females with red-eyed males would result in all of the offspring having white eyes. Does this hypothesis fit with your second cross?
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Look over your data and conclude which of the hypotheses best fits the patterns you found in your crosses. If your data does not fit with any of these, hypothesize why this might be the case.
</div></li></ul></li></ol>

Genetics of Organisms: REF and WEF Fly Genetics - Procedure

REF and WEF Fly Genetics
ExpandNOTE: At your station, you should have four vials of flies. They will be labeled as male or female. They will also be ...

Hardy-Weinberg and Genetic Drift

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Class Simulation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Begin by opening a new spreadsheet file. Following the Hardy-Weinberg equation where p is the frequency of a dominant allele A in a population, and q is defined as the frequency of a recessive allele B, input frequency p of allele A into cell B2, and frequency q of allele B into cell B3.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Assign the value 0.5 to cell C2.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Following the 1 &#8211; p = q equation, enter the formula &#8220;= 1 &#8211; C2&#8221; into cell C3 to calculate the frequency q of allele B. NOTE: Cells C2 and C3 will represent the gene pool used in the next few steps.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Label cells E2 and F2 &#8220;Allele 1&#8221; and &#8220;Allele 2&#8221; respectively, then enter the RAND code for random formula into cell E3. =IF (RAND()</div></li><li data-sub-note-item="1-5"><div class="lab-step">Select cell E3 and drag the bottom right corner of the cell down to E27 to duplicate the formula into 25 cells, creating an allele for the other offspring, and enter the same formula that was used in cell E3 into cell F3.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Add a third column of data starting with the description &#8220;Genotype&#8221; in cell G2, and add the CONCATENATE function to cell G3 to combine the two randomly generated alleles and to create a genotype.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Drag this formula down for 25 cells and label cells H2, I2, and J2 AA, AB, and BB respectively.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Then, input one IF function as shown here into cells H3, I3, and J3. Drag the formulas down for 25 rows per column. The formulas will return a 1 if the genotype in this row matches the genotype in the header row, and a 0 if the genotype in this row is one of the other two genotypes. H3: = IF (G3=&#8221;AA&#8221;,1,0) I3: =IF(OR(G3=&#8221;AB,G3=&#8221;BA&#8221;),1,0) J3: =IF(G3=&#8221;BB&#8221;,1,0)
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, label cell D28 as &#8220;SUM&#8221;, add the formula to cell H28, and drag the formula to cells I28 and J28 to get the total number of each genotype.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Next, label cells H30 and J30 &#8220;A&#8221; and &#8220;B&#8221; respectively and label cell D31 &#8220;Number of alleles&#8221;.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Add code to cells H31 and J31 to obtain the total number of alleles in the simulated generation, which is two times the frequency of the respective homozygous genotype, plus the frequency of the heterozygous genotype.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, label cell E32 &#8220;Next gen allele frequency&#8221; and add code to cells H32 and J32 to obtain the ratio of alleles in the next generation.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Using the gene frequencies generated in the mathematical model you just built, adjust the gene pool values in cells C2 and C3 to those from H32 and J32, and run 10 more generations, updating the gene pool with the resulting allele frequency each time.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Record the ratio of the two variables at each time point in the spreadsheet on the table, and create a line graph to see how a small population changes over time. Hypotheses: The experimental hypothesis in this study is that smaller populations will deviate more from the Hardy-Weinberg expected frequencies of the alleles than will larger populations, because smaller populations are more susceptible to genetic drift. The null hypothesis is that the allele frequencies within the populations will not differ from the Hardy-Weinberg equilibrium equation expected frequencies, meaning that the allele frequencies will be the same from generation to generation.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Set the frequency of gene A to 0.3 and gene B to 0.7, and run the model 10 more times, recording the ratio of the genes in the next generation in the table after each run.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, drag the formulas for the cells further down to alter the model to include 100 zygotes and run the model another 10 times.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">After the last run, save all of the working data to a personal storage device and exit the spreadsheet program.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Simulation for Hardy-Weinberg and Genetic Drift
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To begin, find a partner and then collect one bag from the instructor.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Verify that the bag contains beads of two different colors and an even number of each color. 
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Designate one color as allele capital A and the other as small a. Hypotheses: The experimental hypothesis for this simulation is that allele frequencies will not change over the 10 generations of the experiment, and Hardy-Weinberg equilibrium will be observed. The null hypothesis is that equilibrium will not be seen in the population.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Note the initial frequency in Table 1 next to generation zero. Using the Hardy-Weinberg equation, calculate the initial genotype frequencies of the population and note this in Table 1. <a href="https://www.jove.com/CDNSource/lm/labs/4/Lab_4_Table_A.xlsx" target="__blank" style="display:block;margin:20px;">Click Here to download Table A</a>
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Then, discuss with your partner about the expected final allele and genotype frequencies after 10 generations of Hardy-Weinberg equilibrium. Note this next to prediction in Table 1.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Draw a pair of beads from the bag. This represents the genotype of one individual in the next generation.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Place a tally mark in the appropriate column next to Generation 1.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Now, keeping each pair of beads out of the bag every time, repeat the drawing 20 times to fill out the generation one row of Table 1.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Using the tallies, calculate the allele frequency for that generation, remembering to count both alleles in each genotype. Make a note of the new allele frequency, and then adjust the 100 beads to reflect the population after Generation 1 by adding and removing beads as necessary.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Repeat the drawing of pairs from this adjusted population for another generation of 20 picks, recording all of the observations in Table 1.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">After calculating the allele frequency for this generation, adjust the population again to set up a third generation draw.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Keep drawing, calculating allele frequencies, and adjusting the starting population until 10 generations have been recorded in total.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Violations of Hardy-Weinberg 
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">To test the effect of different physical and ecological scenarios on the Hardy-Weinberg equilibrium, first draw a slip of paper to determine which Hardy-Weinberg assumption you will be testing.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Make a prediction of what you think might happen in your selected scenario before beginning the experiment, and record this on the board.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">If you selected the mutation scenario, select 5 alleles at random from the set and replace them with 5 of a third color, allele capital B.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">As before, perform the simulation by pulling pairs of beads from the bag for a generation of 20 picks, recording the results.  <a href="https://www.jove.com/CDNSource/lm/labs/4/Lab_4_Table_B.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table B</a>
</div></li><li data-sub-note-item="3-5"><div class="lab-step">After each generation, make adjustments to have the starting population match the new allele frequency as before.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Then, replace 5 random alleles again with the new color, and simulate the new generation for 20 picks.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">If you picked the non-random mating test, draw 1 allele at a time instead of 2 and then pair it with another of the same color pulled from the bag.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">As with the previous simulation, adjust the population to match the new allele frequencies at the end of the previous generation, and carry out the simulation for 10 generations in total.
</div></li><li data-sub-note-item="3-9"><div class="lab-step">For the gene flow condition, begin by removing 10 alleles from the population and replacing them with 10 randomly selected alleles from another set of 100 beads.
</div></li><li data-sub-note-item="3-10"><div class="lab-step">Adjust the allele frequency and repeat the gene flow of removing 10 beads and adding 10 beads from another bag at the beginning of each generation.
</div></li><li data-sub-note-item="3-11"><div class="lab-step">If you drew small population size, simply start each generation with 60 alleles instead of 100.
</div></li><li data-sub-note-item="3-12"><div class="lab-step">As usual, adjust allele frequencies each generation and record 10 generations. In the selection condition, draw from the original bag of beads but do not count any homozygous recessive pairs that are drawn when calculating the allele frequency at the end of each generation.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Simulation of Genetic Drift
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">Make your predictions before running the simulations and record them on the board.
</div></li><li data-sub-note-item="4-2"><div class="lab-step">If you have the small population size test, run the standard Hardy-Weinberg simulation as before, but instead start each generation with 30 alleles and not 100.
</div></li><li data-sub-note-item="4-3"><div class="lab-step">If you drew the founder effect with two alleles test, start generation 1 by drawing only 5 pairs of beads from the bag.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Then, creating the gene pool for generation 2, include only 50 beads at the allele frequency determined in generation 1. When you create the gene pool for generation 3 and all subsequent generations, include 100 beads.
</div></li><li data-sub-note-item="4-5"><div class="lab-step">For a founder effect with 10 alleles, you will need to collect a bag containing random amounts of 10 different colored beads.
</div></li><li data-sub-note-item="4-6"><div class="lab-step">Designate an allele name for each of the new alleles, and then calculate their initial allele frequency.
</div></li><li data-sub-note-item="4-7"><div class="lab-step">Now, run the experiment in the same way as the founder effect with two alleles, by drawing just 5 pairs randomly for generation 1 and then creating the second generation with 50 alleles, and third and subsequent generations with 100. <a href="https://www.jove.com/CDNSource/lm/labs/4/Lab_4_Table_C.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table C</a>
</div></li><li data-sub-note-item="4-8"><div class="lab-step">If you have the natural disaster with 2 alleles, you will need to collect a paper plate from your instructor.
</div></li><li data-sub-note-item="4-9"><div class="lab-step">Draw a line down the middle of the plate. Before generation 1, pour out all of the beads from a standard 100 bead setup onto the plate, and mix them randomly, spreading them evenly.
</div></li><li data-sub-note-item="4-10"><div class="lab-step">Choose one side of the plate and take all of the beads from that side. You should draw from these to create generation 1.
</div></li><li data-sub-note-item="4-11"><div class="lab-step">For generation 2 and beyond, adjust the population and use a total of 100 beads.
</div></li><li data-sub-note-item="4-12"><div class="lab-step">Finally, if you selected the natural disaster with 10 alleles test, you will need to collect a bag containing 10 different colored beads.
</div></li><li data-sub-note-item="4-13"><div class="lab-step">Assign a name to each new allele, and then place them on the plate and select half. Draw from these 50 beads for generation 1, and then calculate the allele frequencies to make a population of 100 in each subsequent round.
</div></li></ul></li><li data-note-item="5" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="5-1"><div class="lab-step">Using the entered functions, calculate the number of each allele by multiplying the number of the homozygous genotypes by 2 and adding the number of the heterozygous genotypes.
</div></li><li data-sub-note-item="5-2"><div class="lab-step">The frequency can then be calculated by dividing the number of the allele in question by the total number of alleles. Enter the results into the table.
</div></li><li data-sub-note-item="5-3"><div class="lab-step">Next, use the Hardy-Weinberg equation to calculate the expected genotype frequency of the next generation, and compare the results of the simulation with the expected frequency, using the table as a guide.
</div></li><li data-sub-note-item="5-4"><div class="lab-step">Plot the changes of the population gene frequencies over time that you recorded in the table. Note any changes in allele frequencies over time and if any alleles decrease in frequency to the point that they disappear from the population. 
</div></li><li data-sub-note-item="5-5"><div class="lab-step">Plot the changes in the populations of the 25 and 100 zygotes with initial gene frequencies of 0.5 for each allele. Compare the results of the smaller population and the larger populations.  NOTE: The observed allele frequencies are expected to differ slightly from the Hardy-Weinberg expected values as a result of chance from the RAND function.
</div></li><li data-sub-note-item="5-6"><div class="lab-step">Using separate lines for each allele, graph the allele frequencies found for the original Hardy-Weinberg experiment over 10 generations.
</div></li><li data-sub-note-item="5-7"><div class="lab-step">Compare the results across the class, and see if your results agree.
</div></li><li data-sub-note-item="5-8"><div class="lab-step">Next, again using one line per allele, graph the results of your second experiment testing violations of the Hardy-Weinberg equilibrium, and share your finding with the class. Compare your actual results with your prediction. 
</div></li><li data-sub-note-item="5-9"><div class="lab-step">Finally, graph the results of your genetic drift simulation scenario in the same manner.
</div></li><li data-sub-note-item="5-10"><div class="lab-step">Again, present your results to the group and hypothesize why your results did or did not match your prediction. Compare your results to those of your classmates&#8217;. 
</div></li></ul></li></ol>

Hardy-Weinberg Equilibrium Principle and Genetic Drift - Procedure

Class Simulation
ExpandBegin by opening a new spreadsheet file. Following the Hardy-Weinberg equation where p is the frequency of a dominant allele A in a ...

Animal Diversity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Form and Function of Crickets and Crayfish
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">This lab involves dissection of both crickets and crayfish. You will need one cricket and one crayfish for each pair of students. IMPORTANT: Purchase the largest crickets available and place them in the freezer the night before.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">On the day of the lab if they are frozen, allow the crickets to thaw for at least three hours before the dissections begin.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">While waiting for the crickets to thaw, melt dissecting wax in a microwave or on a hotplate until it is completely liquefied.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Fill enough Petri dishes to have one per student pair to around halfway. Allow these plates to harden at room temperature.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Next set out a dissecting tray, 12 pins, scissors and a needle probe for each pair of students.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Also set up a few dissecting microscopes around the room and turn them on. NOTE: Generally, one microscope for four students is sufficient.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Then, set out a pitcher or beaker of water for students to use during the cricket dissection.
</div></li></ul></li></ol>

Animal Diversity: Form and Function of Crickets and Crayfish - Prep

Form and Function of Crickets and Crayfish
ExpandThis lab involves dissection of both crickets and crayfish. You will need one cricket and one crayfish ...

Microbial and Fungal Diversity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Examining Microbial Colony Structures
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To begin, collect your allocated culture plates and then remove the Parafilm and lid from one of them.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Place the uncovered plate under a dissecting microscope and then observe the individual communities within each quadrant of the plate.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">As the colonies are identified, fill out the observed colony properties in the appropriate column in the table.  <a href="https://www.jove.com/CDNSource/lm/labs/28/Table_1_Microbial_diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then, repeat the observations for each of the other two bacterial species.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">To perform Gram staining of your three bacterial species, first label the ends of three microscope slides with the initials of each bacteria species, your initials, and the date. HYPOTHESES: In this experiment, the experimental hypothesis might be that it is possible to elucidate different bacterial species and shapes using Gram staining, and that Gram-positive bacteria will stain purple with crystal violet whereas Gram-negative bacteria will appear pink due to the safranin counterstain. The null hypothesis could be that all of the bacterial types will retain both stains equally, and staining will not help elucidate their species or shape.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">To begin the first stain, attach a clip to the slide for ease of handling. For each bacterial smear, remember to be sure to use a cleaned and flame-sterilized inoculating loop.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Place two loopfuls of sterile distilled water onto the center of the slide.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">After sterilizing the loop again, cool the loop by touching an uninoculated portion of the agar several times.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, remove a small amount of culture from an isolated colony and mix it with the water on the slide.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Allow the slide to dry at room temperature and heat-fix the smear by quickly passing it through a flame.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Once dry, pipette several drops of crystal violet onto the slide and let it sit for two to three minutes.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Carefully rinse the slide with distilled water by aiming the direct flow towards the top of the slide, allowing the water to gently flow down. Do not aim the water flow directly at the smear.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Next, cover the slide with Gram&#39;s iodine, leave it to sit again for two minutes, and then gently rinse with distilled water.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Decolorize the slide with 95% ethanol until stain no longer washes away.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Immediately, rinse the slide with distilled water. This will limit over-decolorizing.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, add several drops of safranin counterstain and leave for 30 s. This will stain any Gram-negative cells present.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Gently rinse the slide with distilled water and blot dry with absorbent paper.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">After staining the other two slides in the same manner (steps 6 &#8211; 17), observe each with a microscope.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Use a low power objective first to make coarse adjustments. When you find an ideal section, move on to the higher magnifications.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">After further imaging and adjusting the smear with a medium power objective, add immersion oil directly to the smear. NOTE: The oil is needed for high power objectives that will provide the best micrographs.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Now, use colored pencils to draw the observed bacteria in the appropriate magnification circles and fill in the observed characteristics in the appropriate column, noting the bacteria&#39;s shape and whether it is Gram-positive, identified by darker purple staining, or Gram-negative, identified with pink staining. <a href="https://www.jove.com/CDNSource/lm/labs/28/Table_2_Microbial_diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Finally, repeat the observation, drawing, and characterization for the other two stained bacterial slides.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Examining Fungal Structures
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Remove the <em>S. cerevisiae</em> plate from the incubator and place a drop of water onto a microscope slide.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Using a sterilized inoculating loop, smear a film of yeast into the drop of water and place a cover slip over the slide.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Place the slide under the microscope at the lowest magnification, increasing the magnification to the oil immersion setting to see the individual yeast cells.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Observe the general shape and structure of the yeast and identify any cells that are undergoing asexual reproduction by budding.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Now, draw the cells in the appropriate magnification circles.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">When you are done observing the yeast, replace the <em>S. cerevisiae</em> slide with a prepared Ascomycetes apothecium slide and set the microscope to the lowest magnification.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Using colored pencils, draw the structures at low and high magnifications, including the asci and ascospores.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Finally, place a button mushroom, <em>Agaricus bisporus</em>, under the dissecting microscope and draw the features, including the pileus, or cap, the gills, and the stipe, or stalk.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Using the coloring of the bacteria recorded in the table, determine which species are Gram-positive and which are Gram-negative.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Then, look at the shapes recorded in the table to determine which bacterial species are bacillus, coccus, or spirillum.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Next compare your illustrations of the yeast cells to your illustrations of the bacterial cells. NOTE: Yeast from the Ascomycetes phylum, such as <em>S. cerevisiae</em>, can reproduce asexually, which can be observed through the budding of the cells under a microscope.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Record any observations of yeast cells budding.  NOTE: Ascomycetes can also reproduce sexually via mitosis and haploid spore formation.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Record any observations of spore or asci formation you observed in the table.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">NOTE: Fungus from the Basidiomycota phylum exhibit clearly observable gills under the pileus.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Record any fungal species that demonstrated gills under the pileus.
</div></li></ul></li></ol>

Microbial Colony and Fungal Diversity - Procedure

Examining Microbial Colony Structures
ExpandTo begin, collect your allocated culture plates and then remove the Parafilm and lid from one of them.
Place ...

Macromolecules

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Identifying Macromolecules with Indicator Solutions
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">HYPOTHESES: In this activity, the experimental hypothesis might be that the addition of an indicator dye to each test solution will result in a color change if the test solution contains the macromolecule of interest. The null hypothesis might be that the addition of an indicator dye will not result in a color change in any of the test solutions.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Place 16 clean test tubes in a tube rack in four rows of four tubes and label each row A through D and each column 1 - 4.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Label each test tube with its corresponding position in the test tube rack.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">After this, stir solution A thoroughly and use a pipette to add 4 mL of solution A to each tube in Row A.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Next, stir solution B until it is well mixed and use a new pipette to add 4 mL of solution B to each tube in Row B.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">After stirring solution C thoroughly, add 4 mL of solution C to each tube in Row C.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Then add 4 mL of soybean-derived vegetable oil to each tube in Row D.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">When all of the tubes have been filled with their respective test solutions, add 200 mL of water to a 400 mL beaker on a hotplate and set the hotplate to high.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">To perform the Benedict&#39;s Test, add 2 mL of Benedict&#39;s reagent to test tubes A1, B1, C1, and D1 while the water is warming.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Observe the initial color of the tubes.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">When the water begins to boil, transfer each test tube to the beaker and heat the tubes in the boiling water bath for 3 - 5 minutes.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, record the final color of each of the tubes in the appropriate cells of the table and turn off the hotplate. <a href="https://www.jove.com/CDNSource/lm/labs/22/Table_1_Macromolecules.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-13"><div class="lab-step">To perform an Iodine-Potassium-Iodide Test, first observe the color of the solutions in the test tubes in column 2.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Then, add 1 mL of iodine potassium iodide reagent to tubes A2 to D2 and record the final color change in each of the tubes.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">For the Sudan IV Test, observe and record the color of the solutions in the test tubes in column 3.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, add 2 - 3 drops of Sudan IV reagent to test tubes A3 through D3 and record the color change, if any, in each tube.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">For the Biuret Test, observe the color of the solutions in the test tubes in column 4.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Then, add 1 mL of Biuret reagent to test tubes A4 through D4 and record the final color in each of the tubes.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Using the results from the indicator tests, each of the four original solutions can be identified as a sugar, starch, lipid, or protein. Refer to the Concepts video to help you determine which tubes contain which macromolecules.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">First, look at the results from the Benedict&#39;s Test. Determine which of the test solutions contain sugar based on your results.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Next, create a bar graph with your five plant species listed on the x-axis and the surface area in cm indicated on the y-axis.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Next, look at the results from the Iodine-Potassium-Iodide Test. Determine which of the test solutions contain starch.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Now, review the results from the Sudan IV Test. Using these observations, determine if any of the test solutions contain lipids.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Finally, look at the results from the Biuret Test. Determine if any of the test solutions contain proteins.
</div></li></ul></li></ol>

Macromolecules: Identification Using Indicator Solutions - Procedure

Identifying Macromolecules with Indicator Solutions
ExpandHYPOTHESES: In this activity, the experimental hypothesis might be that the addition of an ...