Help! Sequencing 100bp PCR by Automated sequcencer

Dr. Volker Blaschke vblasch at gwdg.de
Wed Aug 27 01:20:52 EST 1997


Dharsha wrote:
> 
> 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

Hi, 

we have sequenced PCR products of ~130 bp in size by automated cycle
sequencing (ABI machine). What we did is use the Qiagen PCR cleanup kit
which gets rid of the primers. The product is the eluted with 30 µl of
H20. Of these, I just took 3 µl, sometimes 1 µl was enough, and it
worked very well. The sequencing kit was from PE, by the way.

Hope this helps,

  Volker



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