GecKO CRISPR Screen PCR#1/#2

[didem tecimel]

Hi everyone,

I am confused about the PCR steps. I will use MGI platform for the sequencing thats why I am not able to use the primers for PCR2 as illumima adaptors are not suitable for me. I plan to do one step PCR and then proceed with the library presentation. I check if gecko library is present in my cells in a small scale so that ı can continue with my experiments.  However, day 7 has sharp bands the band intensity lowers as I try day15 and day19. I think this is common as I see previous discussion in this forum but can somebody inform me about the reason and if I should perform nested PCR before library prep?

Best,

Didem

Didem TECIMEL, M.Sc.

PhD student

Yeditepe University School of Medicine

Department of Medical Genetics

Istanbul / TURKEY