PCR product of the amplified sgRNA library is not enough for NGS to determine sgRNA distribution

After I amplify the pooled sgRNA library, I need to do next-generation sequencing of the amplified sgrna library to determine sgrna distribution.

So, I do the PCR as step 32 in protocol with 20ng template in 50μl reaction system with 10 reactions in total. The cycle number is 22.

I got 500 μl PCR products and do PCR purification and gel extraction.

But, after I finish all procedures as protocol. The PCR products will be only about 40 ng/μl in 30μl. The NGS company inform me the successful rate of NGS is just 60-70% due to insufficient sample amount. I repeat three times. Every time result is similar.

So, I hope to know how about the volume and concentration of your samples for NGS to determine the amplified sgRNA distribution.


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Fuchsia Kelpieabout 2 years ago

Normally for our NGS runs, 40ng/uL in 30uL is a lot more than we need for MiSeq or NextSeq sequencing (about 100 NGS runs). If the NGS company is requesting more sample for sequencing, you can scale up the number of PCR reactions you are running accordingly, ie do 30 reactions instead of 10 reactions.

Green Mummyabout 2 years ago

Thanks so much for your recommendation, and it is so helpful.

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