Name: small-scale extraction of caenorhabditis elegans genomic dna
Uploaded: 2022-06-07T13:00:00
Description: Watch this Scientific Journal Video about Small-Scale Extraction of Caenorhabditis elegans Genomic DNA at JoVE.com

The N2 strain and E. coli OP50 bacteria were obtained from the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The blmp-1(tm548) strain was obtained from the National Bioresource Project, Japan. The authors thank WormBase. This work was supported by NIH R01GM097591 to T.L.G., internal funding by Texas Woman&#39;s University to T.L.G, and TWU&#39;s Center for Student Research to M.F.L.

1. C. elegans maintenance
NOTE: N2 (wild type) and blmp-1(tm548) C. elegans strains were maintained on standard nematode growth media (NGM) plates at 20 &#176;C.
<ol>
	<li>Prepare NGM plates by dissolving 23.005 g of NGM powder in 973 mL of water in a 2 L flask. Cover the flask opening with aluminum foil (or autoclavable cap that allows venting) and secure the covering with autoclave tape. Autoclave to dissolve the powder and sterilize the contents with a s...

<ol>
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Extract-N-Amp Tissue PCR Kit technical bulletin Available from: <a target="_blank" href="https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/249/244/xnat2bul.pdf">https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/249/244/xnat2bul.pdf</a> (2013)</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , (2006).</li><li>Sutphin, G. L., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Caenorhabditis+elegans+life+span+on+solid+media.">Measuring Caenorhabditis elegans life span on solid media.</a> Journal of Visualized Experiments: JoVE. (27), e1152 (2009).</li><li>Chaudhuri, J., Parihar, M., Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+worm+lab:+from+culturing+worms+to+mutagenesis.">An introduction to worm lab: from culturing worms to mutagenesis.</a> Journal of Visualized Experiments: JoVE. (47), e2293 (2011).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> Journal of Visualized Experiments: JoVE. (62), e3923 (2012).</li><li>Gumienny, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+SMA-10/LRIG+is+a+conserved+transmembrane+protein+that+enhances+bone+morphogenetic+protein+signaling.">Caenorhabditis elegans SMA-10/LRIG is a conserved transmembrane protein that enhances bone morphogenetic protein signaling.</a> PLoS Genetics. 6, 1000963 (2010).</li><li>Nelson, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bow-tie+genetic+architecture+for+morphogenesis+suggested+by+a+genome-wide+RNAi+screen+in+Caenorhabditis+elegans.">A bow-tie genetic architecture for morphogenesis suggested by a genome-wide RNAi screen in Caenorhabditis elegans.</a> PLoS Genetics. 7, 1002010 (2011).</li><li>Zhang, L., Zhou, D., Li, S., Jin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLMP-1+contributes+to+collagen-related+morphogenesis+in+C.+elegans.">BLMP-1 contributes to collagen-related morphogenesis in C. elegans.</a> Life Science Journal. 9, 1080-1088 (2012).</li><li>Horn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DRE-1/FBXO11-dependent+degradation+of+BLMP-1/BLIMP-1+governs+C.+elegans+developmental+timing+and+maturation.">DRE-1/FBXO11-dependent degradation of BLMP-1/BLIMP-1 governs C. elegans developmental timing and maturation.</a> Developmental Cell. 28, 697-710 (2014).</li><li>Huang, T. -. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLMP-1/Blimp-1+regulates+the+spatiotemporal+cell+migration+pattern+in+C.+elegans.">BLMP-1/Blimp-1 regulates the spatiotemporal cell migration pattern in C. elegans.</a> PLoS Genetics. 10, 1004428 (2014).</li><li>Lakdawala, M. F., Madhu, B., Faure, L., Vora, M., Padgett, R. W., Gumienny, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+interactions+between+the+DBL-1/BMP-like+pathway+and+dpy+body+size-associated+genes+in+Caenorhabditis+elegans.">Genetic interactions between the DBL-1/BMP-like pathway and dpy body size-associated genes in Caenorhabditis elegans.</a> Molecular Biology of the Cell. 30, 3151-3160 (2019).</li><li>Madeira, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EMBL-EBI+search+and+sequence+analysis+tools+APIs+in+2019.">The EMBL-EBI search and sequence analysis tools APIs in 2019.</a> Nucleic Acids Research. 47, 636-641 (2019).</li></ol>

Determining the genotypes of C. elegans is an important step while performing genetic crosses to create new C. elegans strains. Genomic DNA extraction using a single or few C. elegans is a crucial step in genotyping C. elegans. This protocol describes genomic DNA extraction from C. elegans using a commercial kit. This method is fast and works robustly. The genomic DNA extracted using this method can be used for downstream applications, including genotyping, sequencing, and clo...

Here, two related protocols are presented for the lysis of Caenorhabditis elegans to make DNA accessible for PCR-based applications. PCR is a commonly used molecular technique used for many applications, including genotyping and amplifying DNA fragments for cloning and sequencing, among others. The small (1 mm), free-living roundworm C. elegans is a popular animal system for biological research. Obtaining suitable genomic DNA from a single animal or a few animals is sufficient to amplify the sequence by PCR. Late L4 larvae and adults contain only ~1,000 somatic cells (including some multi-nuclear, polyploid cells), germ cells, and (if the animal is a gravid hermaphrodite) offspring in utero1. However, these animals are protected by a cuticle that must be disrupted to extract the genomic DNA2. Standard methods to prepare nematode genomic DNA template for PCR involve multiple steps and take several hours. The animals are first frozen in worm lysis buffer containing proteinase K (&#8722;70 &#176;C or below) for at least 15-45 min (longer is recommended by some protocols)3,4,5,6. This step cracks open the animals.
After freezing, the animals are incubated for 1 h at 60-65 &#176;C for the proteinase K to work, then the enzyme is inactivated for 15-30 min at 95 &#176;C. The proteinase K destroys the nucleases that degrade DNA. The inactivation of proteinase K before PCR is important to prevent the proteinase K from degrading the DNA polymerase. The two kit-based protocols described here are quick, reliable, and cost-effective methods to extract genomic DNA from either a single animal or a few nematodes for everyday research and teaching laboratory applications. The kit used was originally optimized by the manufacturer to extract DNA from animal tissue, saliva, and hair7. It uses a proprietary tissue preparation solution and extraction solution to lyse cells and make genomic DNA accessible. A proprietary neutralization solution then neutralizes the components that may inhibit PCR (e.g., salts, ions, and Mg2+-binding molecules).
When genotyping, a single animal can be tested. When determining if a strain is homozygous, testing six or more offspring from a single animal gives high confidence that a line is homozygous or not (there is a 0.02% chance of randomly picking six homozygous mutant progeny from a heterozygous parent [(1/4)6 &#215; 100% = 0.02%]). This method 1) is straightforward, with fewer steps than the proteinase K method, and 2) decreases the template preparation time to 15 min. The results in this work demonstrate that the developed protocol works robustly in extracting genomic DNA from single or a few worms, which can be reliably used for downstream applications that do not require highly purified DNA, including PCR.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>autoclave tape</td>
			<td>Defend</td>
			<td>43237-2</td>
			<td></td>
		</tr>
		<tr>
			<td>aluminium foil, heavy duty</td>
			<td>Reynolds Wrap</td>
			<td>2182934</td>
			<td></td>
		</tr>
		<tr>
			<td>calcium chloride</td>
			<td>Millipore Sigma</td>
			<td>102382 (CAS 10035-04-8)</td>
			<td></td>
		</tr>
		<tr>
			<td>Extract-N-Amp kit (includes Tissue Preparation Solution, Extraction Solution, and Neutralization Solution)</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>XNAT2-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp hotplate/stirrer</td>
			<td>Fisher Scientific</td>
			<td>11-100-495H</td>
			<td></td>
		</tr>
		<tr>
			<td>LB media, Lennox, capsules</td>
			<td>MP Biomedicals, LLC</td>
			<td>3002-131</td>
			<td></td>
		</tr>
		<tr>
			<td>magnesium sulfate, 97% pure, anhydrous</td>
			<td>Thermo Scientific</td>
			<td>AC413485000 (CAS 7487-88-9)</td>
			<td></td>
		</tr>
		<tr>
			<td>microcentrifuge</td>
			<td>Labnet International, Inc.</td>
			<td>PrismR, C2500-R</td>
			<td></td>
		</tr>
		<tr>
			<td>NEB Q5U Hot Start High-Fidelity DNA polymerase</td>
			<td>New England Biolabs, Inc.</td>
			<td>M0515S</td>
			<td>&#34;Pol E&#34; used in Supplemental Figure S1, a high-speed, high-fidelity polymerase (20&#8211;30 s/kb)</td>
		</tr>
		<tr>
			<td>NGM media powder</td>
			<td>US Biological Life Sciences</td>
			<td>N1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity PCR Master Mix with HF Buffer</td>
			<td>New England Biolabs, Inc.</td>
			<td>M0531S</td>
			<td>&#34;Pol D&#34; in Figure 1B, a high-speed, high-fidelity polymerase (15&#8211;30 s/kb)</td>
		</tr>
		<tr>
			<td>primers</td>
			<td>Integrated DNA Technologies</td>
			<td>custom DNA oligos</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeSTAR GXL polymerase</td>
			<td>Takara Bio Inc.</td>
			<td>R050B</td>
			<td>&#34;Pol C&#34; in Figure 1B, a high-fidelity polymerase (1 min/kb) for GC-rich templates and templates up to 30 kb</td>
		</tr>
		<tr>
			<td>Quick-Load Purple 2-log DNA Ladder (0.1&#8211;10.0 kb)</td>
			<td>New England Biolabs, Inc.</td>
			<td>N0550S</td>
			<td></td>
		</tr>
		<tr>
			<td>SapphireAmp Fast PCR Master Mix</td>
			<td>Takara Bio Inc.</td>
			<td>RR350A</td>
			<td>&#34;Pol A&#34; in Figure 1B, polymerase used in Figure 1A, C, D, a high-speed polymerase (10 s/kb) for targets up to 5 kb</td>
		</tr>
		<tr>
			<td>Sigma ReadyMix Taq PCR reaction mix</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>P4600</td>
			<td>&#34;Pol B&#34; in Figure 1B, a polymerase (1 min/kb) for targets up to 7 kb</td>
		</tr>
		<tr>
			<td>SimpliAmp thermal cycler</td>
			<td>Applied Biosystems</td>
			<td>A24812</td>
			<td></td>
		</tr>
		<tr>
			<td>stir bar</td>
			<td>Fisher Scientific</td>
			<td>14-512-126</td>
			<td></td>
		</tr>
		<tr>
			<td>vortex mixer</td>
			<td>Fisher Scientific</td>
			<td>2215365</td>
			<td></td>
		</tr>
		<tr>
			<td>worm pick</td>
			<td>Genesee Scientific Corporation</td>
			<td>59-AWP</td>
			<td></td>
		</tr>
	</tbody>
</table>

small-scale extraction of caenorhabditis elegans genomic dna

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Genomic DNA from a single or a few wild-type adults was extracted using the commercial kit or traditional lysis protocol to compare the efficacy of these two methods. These lysates were then used as templates for PCR to amplify either a larger target of ~2,100 bp (encoding blmp-1) or a smaller target of ~500 bp (encoding a part of sma-10). Both methods successfully yielded appropriate PCR products (Figure 1A).
Next, the ability of kit-extracted g...

Watch this Scientific Journal Video about Small-Scale Extraction of Caenorhabditis elegans Genomic DNA at JoVE.com

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and ...

We have developed an easy, fast, and cost-efficient method of extracting C.elegans genomic DNA that works great for classroom and research applications. These protocols can be reliably used to quickly produce DNA templates from a single or a small sample of C.elegance for PCR based applications. Users who do not have experience picking worms or using a micropipetter may struggle with steps requiring these skills.Watching a skilled practitioner and practicing may help. Demonstrating the procedure will be Farhan Lakdawala, a senior graduate student from my laboratory. For extracting DNA from a single Caenorhabditis elegans worm, first, set the lysis program consisting of one cycle at 55 degrees Celsius for 10 minutes followed by 95 degrees Celsius for three minutes in a thermocycler.To prepare the lysis solution, first, add 0.8 microliters of extraction solution from the kit to the inside wall of a 0.2 milliliter PCR tube on ice. Then add 0.2 microliters of tissue preparation solution from the kit to the droplet of extraction solution and mix by by petting. With the selection of a worm for DNA extraction, first, identify an L4 or adult animal under a dissecting microscope.An L4 hermaphrodite can be identified by a characteristic vulva that is visible as a pale half circle in the middle of the animal. To identify an adult hermaphrodite, select one of the largest animals on the plate that may have oval embryos visible in its uterus. Next, using a platinum wire pick, transfer the selected animal to the solution.To collect the contents at the bottom of the tube, centrifuge the tube for two to three seconds at 2000 x g at room temperature. Then place the tube in the thermocycler, and run the lysis program set earlier. After this lysis program is over, briefly centrifuge the tube and put it on ice.Then add 0.8 microliters of neutralization solution from the kit and mix by pipetting. Again, centrifuge the tube for two to three seconds at 2000 x g at room temperature. If the lysate is not used immediately, store the tube at four degrees Celsius for future use.To extract DNA from a individual worms also, first, set the lysis program of one cycle at 55 degrees Celsius for 10 minutes followed by 95 degrees Celsius for three minutes in a thermocycler. Next prepare the lysis solution by first adding two microliters of the extraction solution from the kit onto the inside wall of a 0.2 milliliter PCR tube on ice, followed by the addition of 0.5 microliters of the tissue preparation solution from the kit to the droplet of extraction solution, then mix the contents by pipetting. After identifying L4 or adult animals, use a platinum wire pick to transfer an adequate number of animals into the solution.Next, centrifuge the tube for two to three seconds at 2000 x g at room temperature, then place the tube in the thermocycler and run the previously set lysis program. After completion of the program, briefly centrifuge the tube and place it on ice. Then add two microliters of the neutralization solution from the kit to the tube and mixed by pipetting.Again, centrifuge the tube for two to three seconds at 2000 x g at room temperature. If not used immediately, store the lysate at four degrees Celsius for future use. The genomic DNA extracted by the protocol could be successfully used as a PCR template, both the commercial kit and the traditional proteinase K method produced DNA with similar levels of PCR amplification of a 2100 base pair product and a 500 base pair product.The kit method for DNA lysis also provided PCR templates effective for a variety of DNA polymerase enzymes. Even at different dilutions, the DNA extracted from a single animal by the commercial kit supported the PCR amplification of a 2100 base pair DNA fragment with equivalent efficiency. However, the amplification of a 500 base pair DNA fragment varied with the template concentration.The suitability of the DNA extracted either from a single animal or from multiple animals as a PCR template for determining genotype was further demonstrated by the successful amplification of a gene and its mutated variant. As this protocol involves pipetting small volume of reagents, to ensure consistency, use a master mix for multiple reactions, good piping technique, and well calibrated pipettes. Also, spinning down the contents of the tube ensures proper lysis.This method could be scaled up, adapted to extract C.elegans genomic DNA for other downstream applications, and could be used to extract DNA from other nematode species. Given the simplicity, speed, robustness, and low cost of the protocol, it is well suited for both research and classroom applications where time and resources are limited.

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