I am amplifying the genomic DNA harvested from a small scale CRISPR-ko screen prior to NGS, however I am experiencing issues with the PCR amplification step.
I am following the guidelines provided in the Young 2017 Nature paper (i.e. 1ug genomic DNA, 22 cycles, Tm 63°C), however when I run the full 50ul reaction on agarose gel I cannot visualize any band corresponding to a size of 270-280bp. I have repeated the PCR multiple times on different genomic DNA samples with the same results. I can only see a band at 100bp, which I assume corresponds to the NGS library primers.
I have performed in parallel other PCRs on the same DNA targeting genomic DNA regions, and they were successful. Prior to harvesting of the genomic DNA, the transduced cells were kept under puromycin selection for the entire duration of the screen. The efficacy of the antibiotic selection was tested on an untransduced cell line. Also, a reporter GFP construct in those cells indicate that the assay worked as I could sort green cells.
I am using the same NGS Lib primers as recommended in the Young 2017 protocol. The primers were purchased from IDT as 4nm ultramers and were resuspended to 100uM in nuclease-free water, then diluted to 10uM. I am using a primer concentration of 0.25uM per 50ul reaction. I tested various polymerases (KAPA Hifi, Nebnext, Q5) – still no amplification.
Is it sensible to increase the number of cycles? What else could be the problem?