Hi everyone,
I'm trying some site directed mutagenesis at the moment and having problems with the sequencing. I'm new to sequencing so I thought I'd try to get some tips. I'm currently using Source Bioscience and the T7 promoter as a primer which is 968 base pairs away from the end of the gene I want to sequence. Unfortunately the reads that come back don't stretch that far. My concentration seems good and there's no hint of contamination present. Does anyone have any idea about what could be going wrong? Any tips for sequencing in general would also be greatly appreciated.
Hi, reading length of 900bp are pretty normal for standard sequencing. How far away is the T7 promoter from the start of the gene (and not the end)? How long is the gene? You can always create an internal sequencing primer if neither T7 or T7terminator primer are likely to cover the mutation location (if you have a very long gene). The T7term primer produces reverse compliment readings. Hope this helps!
Even if you were able to read this far, the sequence at that distance would not be great quality. Design a new primer based on the sequence you obtained. If you design your primer at 500-600 bp in the current sequence, this will allow you to read well past the position of interest.
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