ABI sequencing of GC-rich promoter: help!

Bruce Roe broe at aardvark.ucs.uoknor.edu
Thu May 25 07:26:00 EST 1995


In article <Pine.AUX.3.91.950523104807.12782A-100000 at mail.med.cornell.edu>, hmmoss at MAIL.MED.CORNELL.EDU (Heidi Moss) writes...
>I am attempting to characterize a TATA-less GC-rich promoter element, but 
>my sequence data in that region is far from perfect. I know my primers 
>are fine (I am simply working with T7, 22-mer, and T3, 20-mer, for 
>subclones in pBlue, just to be safe for now), and they have worked 
>beautifully in other regions from my genomic subclones. In addition, I 
>carefully purify my DNA via Qiagen and quantitate by spec and agarose gel. 
>Nonetheless, this region proves to be troublesome. 
>Any advice would be appreciated: general or specific!!
>thanks!
>-- Heidi
> 
Heidi,
	We have had our best success in seuqencing through similar
really GC-rich regions using the Taq-terminators on the ABI.  Although
I dislike using "kits", in this instance, the ABI Taq-terminator Kit
and their protocol gave very satisfactory results when we added
distilled DMSO to a final concentration of 10%.  Although I have
no direct evidence, I believe that their inclusion of dITP instead
of dGTP in the reaction mixes is one of the factors that results in
improved read through GC-rich regions using their "kit". I'd suggest you
give their kit a try and titrate in distilled DMSO.

Cheers.......bruce
Disclamier - ABI gives me nothing but a hard time.  :-)
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