double-stranded sequencing GC-rich inserts

USA::RGB12955 RGB12955%USA.decnet at USAV01.GLAXO.COM
Thu Jul 16 10:52:00 EST 1992


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

More information about the Methods mailing list