I have some troubles in clone my custom library, but I'm not sure what's wrong.
My custom library contains 12000 sgRNAs, which is small. but when we performed the NGS after plasmids extraction, it showed that only 1000 sgRNA was detected, and among the 1000 sgRNA, 300 sgRNA was abundant while others was only 1~2 copy.
1. We performed 26 cycle in PCR amplification of pooled oligo library with NEBnext, the protocol suggest less than 20 cycles, but the PCR product is weak when use 20cycle. I wonder if the 26 cycle indroduced the biases？
2. The protocol suggest "After the reaction is complete, pool the PCR reactions and purify the PCR product using the QIAquick PCR Purification Kit, then Run the PCR-purified oligo library from Step 5 on a gel , after that Gel-extract the purified PCR product using the QIAquick Gel Extraction Kit" but we performed gel-extract the PCR product without do PCR Purification, does it matters?
3.Gibson assembly was done according the protocol.