Help! Sequencing 100bp PCR by Automated sequcencer

Dharsha 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 
right track.

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 
size.  

Thanks

Dharsha



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