Help! Sequencing 100bp PCR by Automated sequcencer
mmm_dthurai at mednov1.auckland.ac.nz
Wed Aug 27 16:45:18 EST 1997
I have a ~120bp PCR product whcih I am trying to sequence.
I am taking the PCR product, purifying using BM HiPCR product
purification kit, checked the concentration using a DNA mass ladder.
I am using a 20mer (tm 64.2 C by %GC) and a reverse primer 16bp (tm
64.9 C by %GC). I have sent primer, and 10ng/ul (80 ng) of the
template. I have also tried 5ng/uL template.
I had some partial sequence and when I blastn etc using the GCG and
blast programs I got a match with IgH, meaning I was at least on the
Problem is is that partial sequecne is the best I can get, and tehre
is no complimentarity between my two strands.
Does anyone have any ideas of what to try. Have people tried
sequencing a product this small by automated sequenceing-or should I
try this with manual sequencing? In any case I would really
appreciate some tips for directly sequencing a PCR product of this
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