ds DNA sequencing surefire protocol wanted.

J.J. Baker 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


Jon




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