Hi, I am trying to do editing via crispr cas9 in cancer cell lines. Since I am looking for a very rare event (HR), I should do clone dilution and after screening with ddPCR. I was wondering if the DNA extraction step could be facilitated without going throw DNA extraction kit. Do you have any experience with DNA coming from extraction buffer used in ddPCR? Thanks in advance
In contrast to qPCR where any inhibition lowers the amplification efficiency and thereby makes the Ct-values erroneous relative to the standards, ddPCR involves measurement of the PCR in the plateau phase. Thus, if the generation of the PCR product has high efficiency and makes the product at relative early cycles or the amplification needs e.g. 35x cycles due to (partial) inhibition, this has no influence on the absolute quantification of the DNA in the droplets.
If you have many samples, maybe you should try the non-expensive Chelex resin (Walsh PS, Metzger DA, Higuchi R Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques. 1991 Apr;10(4):506-13).
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