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 sterilization cycle of at least 30 min.</li>
	<li>Cool the medium to 60 &#176;C in a water bath.</li>
	<li>To the cooled medium, add 25 mL of sterile 1 M phosphate (KH2PO4) buffer, pH 6.0; 1 mL of 1 M calcium chloride solution; and 1 mL of 1 M magnesium sulfate solution.</li>
	<li>Stir the medium on a magnetic stir plate to obtain a homogeneous mixture and to prevent uneven cooling and solidification (~5 min). Ensure that the stir bar spins fast enough to create a vortex but not so fast that bubbles are introduced (~250-300 rpm).</li>
	<li>Pour ~25 mL of the medium into each 10 cm Petri plate (~40 plates in total). Dry the plates at room temperature overnight (typically 16-25 &#176;C).</li>
	<li>Grow a colony of Escherichia coli OP50 in 30-50 mL of LB broth overnight at 37 &#176;C.</li>
	<li>Seed each plate with 500 &#181;L of E. coli OP50 culture and let them dry at room temperature before use (typically 16-25 &#176;C)8. Store the plates at 4 &#176;C for up to 3 weeks.</li>
	<li>Transfer C. elegans to seeded plates using a wire pick or other method and let them grow to L4 or adult stage at the temperature required for the strain (typically 16-25 &#176;C, 1-2 days if animals were plated starved as dauers, 3-4 days if animals were plated as embryos)8.</li>
</ol>
2. Single-worm DNA extraction
NOTE: This method is useful for extracting DNA from a single worm (one worm in 1.8 &#181;L of total volume). A master mix can be made if multiple worms will be lysed at one time.
<ol>
	<li>Program a thermocycler for one cycle at 55 &#176;C for 10 min followed by 95 &#176;C for 3 min (lysis program).</li>
	<li>Aliquot 0.8 &#181;L of the extraction solution from the kit onto the inside wall of a 0.2 mL PCR tube on ice. Add 0.2 &#181;L of the tissue preparation solution from the kit to the droplet of the extraction solution. Mix by pipetting.</li>
	<li>Identify an L4 or adult animal under a dissecting microscope9. To identify L4 hermaphrodites, look for a characteristic vulva that is visible as a pale half-circle in the middle of the animal. Identify adult hermaphrodites by looking for the largest animals on the plate that may have oval embryos visible in their uterus.</li>
	<li>Using a platinum wire pick, transfer the selected animal into the solution10.</li>
	<li>Centrifuge the tube briefly (2-3 s, &#8804;6,000 rpm/2,000 &#215; g) at room temperature to collect the contents at the bottom of the tube.</li>
	<li>Place the tube in the thermocycler and run the lysis program set at Step 2.1.</li>
	<li>When the program is complete, briefly centrifuge the tube and place it on ice. Add 0.8 &#181;L of the neutralization solution from the kit to the mixture. Mix by pipetting.</li>
	<li>Centrifuge the tube briefly at room temperature (2-3 s, &#8804;6,000 rpm/2,000 &#215; g).</li>
	<li>Directly use the lysate for PCR or store it at 4 &#176;C or &#8722;20 &#176;C for future use. 
	&#8203;NOTE: Extracted DNA is stable at 4 &#176;C or &#8722;20 &#176;C for at least 6 months7.</li>
</ol>
3. DNA extraction of a few individuals
NOTE: This method is useful for extracting DNA from a few worms. A master mix can be prepared if multiple strains will be lysed at one time.
<ol>
	<li>Program the thermocycler for one cycle at 55 &#176;C for 10 min followed by 95 &#176;C for 3 min (lysis program).</li>
	<li>Aliquot 2.0 &#181;L of the extraction solution from the kit onto the inside wall of a 0.2 mL PCR tube on ice. Add 0.5 &#181;L of the tissue preparation solution from the kit to the droplet of extraction solution. Mix by pipetting.</li>
	<li>Identify L4 or adult animals under a dissecting microscope9. L4 hermaphrodites have a characteristic vulva that is visible as a pale half-circle in the middle of the animal. Adult hermaphrodites are the largest animals on the plate and may have oval embryos visible in their uterus.</li>
	<li>Using a platinum wire pick, transfer a few animals into the solution: 9 animals yield 1 animal equivalent of genomic DNA per 0.5 &#181;L of lysate, and 16 animals yield ~1 animal equivalent of genomic DNA in 0.25 &#181;L (3.5 nematodes/&#181;L)10. 
	NOTE: It is difficult to transfer greater than 16 animals into this volume.</li>
	<li>Centrifuge the tube briefly at room temperature (2-3 s, &#8804;6,000 rpm/2,000 &#215; g).</li>
	<li>Place the tube in the thermocycler and run the lysis program set at Step 3.1.</li>
	<li>When the program is complete, briefly centrifuge the tube and place it on ice. Add 2 &#181;L of the neutralization solution from the kit to the mixture. Mix by pipetting.</li>
	<li>Centrifuge the tube briefly at room temperature (2-3 s, &#8804;6,000 rpm/2,000 &#215; g).</li>
	<li>Directly use the lysis for PCR or store it at 4 &#176;C or &#8722;20 &#176;C for future use. 
	&#8203;NOTE: Extracted DNA is stable at 4 &#176;C or &#8722;20 &#176;C for at least 6 months7.</li>
</ol>
4. PCR reaction
NOTE: One downstream application of this worm lysis technique, detecting a deletion mutation using a fast polymerase, is described. The effectiveness of the two worm lysis protocols in producing genomic template DNA for successful PCR at dilutions to 1/50th of a worm per reaction is also demonstrated.
<ol>
	<li>Extract DNA from a single worm and 16 worms using wild-type N2 and mutant strains, as described above in Step 2 and Step 3.</li>
	<li>Prepare 2-, 10-, 20-, and 50-fold dilutions of single-worm DNA from wild-type N2 animals.</li>
	<li>Set up PCR reactions following the manufacturer&#39;s protocol using primers specific to the sequence of interest and hot-start PCR master mix (Table 1 and Table 2). Use 1 &#181;L of undiluted DNA or 2 &#181;L of diluted DNA as the template in each reaction.</li>
	<li>Confirm the PCR products by gel electrophoresis and imaging11.</li>
</ol>

<ol>
	<li>Corsi, A. K., Wightman, B., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transparent+window+into+biology:+A+on+Caenorhabditis+elegans.">A transparent window into biology: A on Caenorhabditis elegans.</a> WormBook. , 1-31 (2015).</li><li>Page, A. P., Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cuticle.">The cuticle.</a> WormBook. , (2007).</li><li>Williams, B. D., Schrank, B., Huynh, C., Shownkeen, R., Waterston, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+mapping+system+in+Caenorhabditis+elegans+based+on+polymorphic+sequence-tagged+sites.">A genetic mapping system in Caenorhabditis elegans based on polymorphic sequence-tagged sites.</a> Genetics. 131, 609-624 (1992).</li><li>Lissemore, J. L., Lackner, L. L., Fedoriw, G. D., De Stasio, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Caenorhabditis+elegans+genomic+DNA+and+detection+of+deletions+in+the+unc-93+gene+using+PCR.">Isolation of Caenorhabditis elegans genomic DNA and detection of deletions in the unc-93 gene using PCR.</a> Biochemistry and Molecular Biology Education. 33, 219-226 (2005).</li><li>Fay, D., Bender, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNPs:+introduction+and+two-point+mapping.">SNPs: introduction and two-point mapping.</a> WormBook. , 1-10 (2008).</li><li>Biron, D., Haspel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans: Methods and Applications. , (2015).</li><li>. 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>

The authors have no conflicts of interest to disclose.

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 ...

small-scale-extraction-of-caenorhabditis-elegans-genomic-dna

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4.9K Views.  Texas Woman’s University. 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.

Video: Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Isolation of Genomic DNA from Mouse Tails

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...