Brown Siren
posted about 5 years ago

Plasmids undergoing NHEJ?

I am trying to screen for gRNA's targeting a plasmid that is being co-transfected into HEK 293T cells. However, when I sequence around the cut site for TIDE, I am not seeing any detectable indel formations.

Has anyone done this before? Can plasmids undergo NHEJ, or is it that the plasmids undergo degradation after cutting?


Fuchsia Bogreabout 5 years ago

I've done this before. We noticed that you can do a T7E assay, but the yield was highly variable (and even from the same sample it was PCR dependent) and it seemed like a large portion of the plasmid was being degraded instead of repaired.


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