ABI PRISM BigDye SEQ problem!!!

Penny M Avoles avoles at PRAIRIE.NODAK.EDU
Sun Feb 7 14:53:41 EST 1999


Hi Mart
	I had very similar problems with the BigDye Kit.  I could only get
reads of about 200 bp with lots of "N's".  I finally used
the conditions I typically used for reactions when I want to
amplify from my plasmid.  I got it to work!  I recieved a run of over
900 bp.  As of yet I haven't repeated
it with other plasmids but you might want to give it a try.  I used 50 ng
of DNA and 1 uM of T7 primer then followed the PE protocal of
amplification conditions.  
Good Luck
Penny


On 6 Feb 1999, Mart Speek wrote:

> Hi SEQUENCERS!
> 
> I am having a problem when using T3 & T7 primers (20-22 mers with
> Tm ~55-65 degrees centigrade) with ABI Prism BigDye Terminator Cycle
> Sequencing Ready Reaction Kit for sequencing plasmid DNA (KS) by running
> standard PCR with annealing temperature 50 degrees (recommended by P-E)
> and ABI 310 machine. Don't get any sequence!
> 
> Has anyone seen similar problem before?
> 
> In fact, I did get a good sequence with these primers when using
> a dRhodamine Kit from the same manufacturer.
> 
> I cannot see any good reason why BigDye should not-to-allow
> to get sequence with AmpliTaq DNA polymerase FS and T3 & T7.
> 
> Meanwhile I am going to reduce the annealing temperature in
> PCR reaction to see whether this has some effect. I'll post
> the results if there is some follow up.
> 
> Mart Speek (ESTONIA)
> 
> 
> 




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