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.
