PCR of a single or a few nematodes is a standard protocol for many C. elegans laboratories. The standard method to prepare genomic DNA template for PCR is to freeze animals in worm lysis buffer containing Proteinase K in an ultralow freezer (-70°C – 80°C) for at least 15 to 45 minutes (longer is recommended by some protocols) (Williams et al., 1992, Fay and Bender, 2008, Biron and Haspel, 2015). This is followed by a one-hour incubation at 60°C – 65°C for the Proteinase K to work, followed by 15-30 minutes at 95°C to inactivate the enzyme activity.We have adapted the Sigma Extract-N Amp kit for use with C. elegans (XNAT2 kit, http://www.sigmaaldrich.com/catalog/product/sigma/xnat2?lang=en&region=US).  Although the instructions in the kit provide directions for preparing C. elegans DNA, using “1 C. elegans in solution” in 225 µL total solution volume, we modified this kit protocol to prepare single animals for PCR analysis and to genotype lines using PCR (Weber and Douglas, 2016). Our method 1) decreases the template preparation time to 15 minutes, 2) decreases the total volume needed to extract the DNA from one animal over 100-fold from the volume specified in the kit instructions, and 3) is at least two times less expensive than the Proteinase K method. Although the cost depends on the amount ordered and the supplier, our method using the Sigma kit costs about $0.007 per reaction, whereas Proteinase K alone costs approximately $0.015- $0.02 per reaction.

Protocol 1: Single worm DNA extraction using the Sigma Extract-N-Amp kit

All steps are done at room temperature.

Start a thermocycler program to heat to 55°C (hold), 55°C for 10 minutes, then 95°C for 3 minutes.

Aliquot 0.8 µL Extraction Solution into a PCR tube. Add 0.2 µL Tissue Preparation Solution. Mix by pipetting.

Pick 1 gravid adult animal into the solution.

Centrifuge the tube briefly (2-3 seconds, ≤ 6000 rpm/2,000xg).

Put the tube in the thermocycler and continue the program (55°C for 10 minutes, 95°C for 3 minutes). When the program is complete, centrifuge the tube briefly.

Add 0.8 µL Neutralization Solution to the mixture and mix by pipetting. The extract can be used for PCR directly or can be stored at 4°C for future use.

Samples will be ready from worm pick to PCR within 15 minutes.

 

Protocol 2: DNA extraction of a few individuals

This method is useful for genotyping crosses, for example, or if a little more than a single animal’s DNA is needed. This yields a final concentration of 3.5 nematodes/µL. 1 µL of this template usually works for our 25 µL PCRs, but optimization of template concentration may be required. This protocol yields enough template (at 1 µL/reaction) for four reactions. With 16 animals’ genomic DNA mixed together, this template is certain to accurately genotype a line (barring fickle primers that preferentially amplify a smaller band or other technical issues).

All steps are done at room temperature.

Start a thermocycler program to heat to 55°C (hold), 55°C for 10 minutes, then 95°C for 3 minutes.

Aliquot 2.0 µL Extraction Solution in a PCR tube. Add 0.5 µL Tissue Preparation Solution to it. Mix the solutions by pipetting.

Pick 16 gravid adult animals into the solution in a PCR tube.

Centrifuge the tube briefly (2-3 seconds, ≤ 6000 rpm/2,000xg).

Put the tube in the thermocycler and continue the program (55°C for 10 minutes, 95°C for 3 minutes). When the program is complete, centrifuge the tube briefly.

Add 2.0 µL Neutralization Solution to the mixture and mix by pipetting. The extract can be used for PCR directly or can be stored at 4°C for future use.

Samples are ready for PCR within 15 minutes.

We found that SapphireAmp Fast PCR Master Mix (Takara Bio USA, Inc, Mountain View, CA, USA) further reduces total time and works more reliably than the PCR Reaction Mix provided in the Extract-N-Amp XNAT2 kit.