GeCKO PCRs after selection screen

I am following the protocol as published in the 2017 science paper (Nature protocols, Zhang lab).

I've successfully amplified and sequenced the library to confirm coverage and gone on to perform my CRISPR experiments. I am now at the post experiment PCR amplification stage. Having quantified my gDNA and set up the appropriate number of PCRS for the amount of gDNA I have I am not particularly happy about the yield post PCR and want to check this is normal, having run on the D1000 on the tapestation I have a yield of 1.47ng/ul which equates to 8.59nM. I will remeasure yield on the Qubit which I haven't done yet so it may come out a bit higher when I do that.

As it says in the Science paper I am doing just 1 round of PCRs (not the 2 stages of PCRS that previous protocols had) and only 14 cycles so I guess I shouldn't expect a high yield. I do get a band of the correct size (approx 265bp) when I run on the tape station, but the yield is low despite pooling many PCR reactions using the Zymo clean up kit with reservoirs. I have pooled 32X 50ul PCR reactions per clean up column and eluting in 150ul, and they have been set up using the various staggered primers for diversity.

My final conc. will be enough to sequence on the Nextseq, I am just wondering now whether I am in fact doing enough cycles andwhether it is normal to have such a low yield at this stage. I am just worried that I am not amplifying enough of my template I guess.

Any thoughts much appreciated.

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Fuchsia Kelpieover 4 years ago

Generally the number of cycles that you need to do is determined by whether you can see your target band on a gel and the absence of the primer dimer. I normally do 22 cycles of PCR and get a much higher yield than you do, but I would say that if you see your target band of the correct size at 14 cycles and your concentration is high enough to sequence, you can probably proceed to NGS.

Yellow Valkyrieover 4 years ago

Thanks for getting back to me. I don't usually see a band on a normal gel after 14 cycles, just when I run on the tapestation which is much more sensitive. Its good to know the number of cycles you usually perform and see how much higher it is. I'll probably up the number I do for future runs to make sure I have a good PCR yield.

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