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