The question is based on Sanger sequencing results.
Gene size is 717bp which falls within the Sanger range.
I was curious as to why did one of the reactions of the same sample show odd results while other primer was the perfect match?
Can you attach original .abi sequence files F and R so that we can see the detailed sequence please?
Paul Rutland
Group1S2T7Promoter reaction worked fine however Group1S2T7Terminator reaction didn't.
Is this sequenced out of plasmid or pcr product. If pcr product what method of primer removal are you using or is the template sent commercially for sequencing please?
Paul Rutland the plasmids pet28a-cerulean were extracted and sent for sequencing. I asked sequencing facility to sequence the cerulean gene using T7 universal primers.
Thank you Yasha N. B. ,
The sequences do help but I am not sure about the cause of the problem.
It is not just a few mismatches in sequence E4 but an entire sequence which has multiple mismatches. This is usually caused by low signal strength and a high background but this is not the case with e4 as the signal strength is quite good but there is a lot of another sequence underlying the proper signal of e4. This is sometimes caused by partly degraded primer but there are too few good peaks for this to be likely. If the primer bound in2 places you could get a similar effect but it is not likely that there are 2 T7 terminator sites.
If you had a small indel close to the T7 terminator primer the sequence could be this messy while the sequence from the other end would still look good and this may be possible. The good sequence from the other end suggests that the amount and quality of the template and the sequencing reagents is good. This would need the plasmid to have both sequences cloned but is possible.
The aspect that I do not like about this possibility is that the shape and general quality of the underlying sequence is poor.
It might be useful to contact the sequence service provider and ask them if capillary E04 on this machine is having blockage or coating problems in other recent runs.
Thank you so much. I think this explains why I saw two different results for the same gene using different primers. I think that there was something wrong with their primer. I will try to contact the facility for the same.
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