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