I performed site directed mutagenesis, transformation, and then I sent out plasmids for Sanger sequencing and found out that there is extension of DNA just before the stop codon. I am not sure where this is coming from. Was it PCR or is it something that is because of sequencing error? If this is because of PCR, what could be the reasons why it happened and how to avoid it? Thanks! (Please see the image attached to the question)
The entire sequence is correct. I also saw the mutation I wanted. Only problem is at 3' end just before stop codon.
Can we see an original .abi sequencing file please?
It is hard to say anything about sequencing from a text file. For instance the intensities of the signal that you are querying may be low due to reagent depletion and the base calls may be spurious but without a sequence file that is guesswork
If I read the pink sequence correctly, there is a cutting site CTCGAG followed by the His6-tag and a stop codon (TGA), so anyhing downstream of that should not matter, unless you did not want the His-tag there. It's not clear what the real problem is, to be honest. As mentioned above, a real sequencing file would help as well.
As Hüseyin Besir says it should not matter but the quality and intensity of the sequence suggests that it is real sequence not artifact. Blastin the next 170 bases gives good homology with Glia maturation factor gene and a variety of cloning vector and expression vector sequences like pET28 and pmET32 and others if this helps to identify why the sequence exists where it is
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