Single Worm PCR: A Method to Extract and Amplify Genomic DNA

– For single worm PCR analysis, allow a hermaphrodite worm に self fertilize and lay eggs that will produce genetically identical offspring and therefore, preserve a copy of the modified strain. Then, transfer the parent に a tube containing worm lysis buffer.

Freeze the tube at minus 80 degrees Celsius に break open the cuticle or outer layer. Heat the sample に plus 60 degrees Celsius に lyse the worm cells releasing the intracellular proteins along with the cell’s genomic DNA. At this temperature, the proteinase K within the buffer degrades the proteins, particularly nucleases that would otherwise destroy the genomic DNA.

Then, raise the temperature に 95 degrees Celsius に inactivate the enzyme. Finally, add the cell lysate に a tube with a PCR master mix composed of reaction buffer, dNTPs, forward and reverse primers for the region of interest, taq polymerase, and water bringing the reaction に a desired final volume. To perform the PCR, run the appropriate thermal cycler program that amplifies the DNA region of interest.

In the following example, we will see a PCR procedure に screen for CRISPR-Cas9 editing events で the genomic DNA of single F1 worms.

– After 1 に 2 days of egg-laying, use a worm pic に transfer the F1s into individual PCR strip tube caps containing 7 microliters of warm lysis buffer. Centrifuge the PCR tubes at maximum speed and room temperature for one minute に bring the animals に the bottom of the tube. Then, freeze the tubes at negative 80 degrees Celsius for one hour.

Next, lyse frozen worms で a thermal cycler using the following program. Then, set up a PCR master mix as shown で this table.

Add 21 microliters of PCR master mix into clean PCR tubes. Then, add 4 microliters of the worm lysis tube and mix well by pipetting.

Then, run the PCR program following the manufacturer’s guidelines.