double-stranded sequencing GC-rich inserts

Bruce Roe broe at aardvark.ucs.uoknor.edu
Fri Jul 17 08:39:00 EST 1992


In article <9207161452.AA21035 at genbank.bio.net>, RGB12955%USA.decnet at USAV01.GLAXO.COM ("USA::RGB12955") writes...
>Netters:
> 
>We've been trying to double-stranded sequence Bluescript inserts from 
>a cDNA that is about 68 - 70% G+C (Sequenase version 2.0 kit, 
>using Stratagene's -20 and reverse primers) with extremely mixed 
>results.  Mostly what we've been seeing is either no sequence at all or 
>darkly smeared lanes, depending on the insert and the primer.  We 
>suspect that the annealing conditions are to blame, as the DNA is 
>clean (Magic-mini) and the primers are new, plus we've gotten it to 
>work okay for one template.  USB said that linearized DNA would 
>make any secondary structure/annealing problems worse, and 
>suggested doing the extensions on ice and the termination reactions at 
>50C.  It didn't solve the problem, and only gave very faint sequences.  
>Does anyone have any suggestions?
> 
>Rich Buckholz/Mary Martinson
>Glaxo Research Institute
> 
Try either Taq-cycle sequencing with end labeled primers or Bst Polymerase.
We've has our best results from PE/Cetus Amplitaq(tm) and BioRad's Bst.
Sorry but we make our own reagents (buffers,d/ddNTPs, etc) so I can't
tell you much about kits although they are available for both enzymes.
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 \  Bruce A. Roe                     Dept. Chemistry and Biochemistry /
 /  BROE at aardvark.ucs.uoknor.edu     University of Oklahoma           \
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