ds DNA sequencing surefire protocol wanted.
mbjjb at s-crim1.dl.ac.uk
Tue Nov 29 11:44:46 EST 1994
: > As the heading says it all, I'm in need of a double stranded DNA
: > sequencing protocol for DNA prepared using one of the diatomaceous earth
: > methods, that gives bands as good as M13 sequencing (or am I asking too much
: > in the quality of sequencing bands).
: > Thanks in advance.
: > Jaz
After many hassles sequencing DNA with proteins tightly
bound, I have a few golden rules;
1: of all the DNA cleanup methods, CsCl/EtBr centrifugation
is definitely the best. For minipreps, rapid boiling is
better than alkaline lysis.
2: try lowering the primer extension temperature to less
than 20 degrees
3: before loading the gel, heat denature for 3 minutes at
95 degrees and immediately put on ice. Always run fresh
gels I have found a difference between fresh gels and
those stored overnight
I hope these suggestions are of some help
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