After I have cloned my gRNAs into the lentiCRISPRv2 vector and sent the construct for sequencing, I have noticed that a single mismatch between my clone and original sequence has been introduced. My gRNA sequence has successfully been cloned in. However, I am concerned about the position of the mismatch since it is located at the beginning of the U6 promoter. I am nor sure whether this would work (It is a missense mutation resulting in AA change)?
FYI: I have been transforming 4 ul of ligation reaction into 50 ul of Stbl3 bacteria, recovering the bacteria for 1 h and 30 minutes at 30 degrees, incubating at 30 degrees for 20-22 hours (Amp conc: 100 ug/ml). On the next day, I have picked up clones for miniprep (5 ml) and used around 300 ul of the overnight culture with 100 ml of LB media+Amp for midiprep, after which I have sent in for sequencing. I sent 2 different clones, but both of them had the mutation at the same position.
Since you have the same mutation in different clones in a region outside the part you manipulated it is likely the change already existed in your original plasmid.
A single nt change to a promoter region is not likely to have a major effect. Since a promoter is not translated (or transcribed) there is no AA change.
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