Hi guys

I have a question that I would be grateful if you could help with.

I did a nucleofection experiment ( LCL cells) and for one sample, I used 2 gRNAs (called 9 and 10 here). After DNA purification and PCR, I saw 2 bands on the gel; I assume the lower band meant some sequence was deleted. This was confirmed by sanger, and I did not get an ICE analysis for this one.

However, I got an ICE analysis for the higher band, but the results do not make much sense. For instance, it shows 95% indels? This means the sequence has 95% indel in it which is very high.

Some parts are entirely deleted, ie, referring to lines 6 and 7. This is not an indel change anymore. I understand the model fit is low but what is the best way to interpret the result when 2 gRNAs are used? I read the ICE knockout protocol and could not find anything there. I have attached my result for this particular sample and I would be grateful if you could let me know what the best way to interpret my results is? If necessary, I can send you the sanger results as well.

Please find the attached file.

Thanks

Melika