I nucleofected iPSCs from one patient line, grew colonies and attempted to genotype them. i Had previously run a PCR gradient to determine the best Tm for the primers. most of the PCR products have multiple bands. Loaded samples with 6x dye w/o SDS.
i did not purify PCr product prior to digest, but ive never Needed to as our digest protocol typically dilutes out the PCR buffer and the gels are usually fine.
for the digest, I incubated at 37c 15min and inactivated at 65c for 20min, as per the instructions on NEB. I loaded samples with 6x dye w/o SDS. The bands for the digest appear larger/smeared. I ran the digest of each colony next to its PCR product.
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