Issues with PCR for NGS library preparation

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?

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Fuchsia Kelpieover 3 years ago

I would recommend the following troubleshooting steps:

1. Test the PCR using 20ng of library plasmid DNA

2. Try different amounts of genomic DNA (0.5, 1, 2, 3ug) with different numbers of PCR cycles (22, 26, 30 cycles) and see if you et a product.

Hope this helps!

Jade Dhampirabout 3 years ago

I'm wondering if you sorted the issue.

Rose Ghoulabout 3 years ago

Yes we did end up solving that issue.
We did a PCR on the library plasmid and could see a band of the appropriate size on the gel.
Then we did a qPCR with some genomic DNA from our sample (10ng) over 45 cycles to determine at which cycle we could get maximum amplification. I think we ended up using 25 cycles per reaction with 1 ug of DNA and that worked nicely.

Hope this helps,

Jade Dhampirabout 3 years ago

I really thank you for your information.

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