Single Worm PCR: A Method to Extract and Amplify Genomic DNA
Trascrizione
– For single worm PCR analysis, allow a hermaphrodite worm a self fertilize and lay eggs that will produce genetically identical offspring and therefore, preserve a copy of the modified strain. Then, transfer the parent a a tube containing worm lysis buffer.
Freeze the tube at minus 80 degrees Celsius a break open the cuticle or outer layer. Heat the sample a plus 60 degrees Celsius a 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 a 95 degrees Celsius a inactivate the enzyme. Finally, add the cell lysate a 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 a 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 a screen for CRISPR-Cas9 editing events di the genomic DNA of single F1 worms.
– After 1 a 2 days of egg-laying, use a worm pic a 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 a bring the animals a the bottom of the tube. Then, freeze the tubes at negative 80 degrees Celsius for one hour.
Next, lyse frozen worms di a thermal cycler using the following program. Then, set up a PCR master mix as shown di 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.
