Strand separation with NaOH

Johan deBoer jdboer at SOL.UVIC.CA
Thu Aug 6 18:27:50 EST 1992


Hi fellow netters,
I just got a question about double stranded sequencing from a collegue.
He is sequencing a fragment (double stranded) with a high GC content.
Denaturation is at 37C for 30 minutes. Sequencing reaction is with Sequenase.
He is worried whether the denaturation will be complete and wants 
to extend the 30 minutes incubation with NaOH for 45 or 60 minutes.

My question is, wouldn't 30 minutes be enough already, or would it
indeed be an improvement.
Any comments would be appreciated.

J.deBoer
sol.uvic.ca (Univ. Victoria)



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