IUBio

Sequencing through GC rich regions

Jim Jim
Wed Dec 11 08:50:07 EST 1996


Akaline denaturation of the DNA prior to adding it to the reaction will
increase the read length considerablely in gc rich regions .Use a x5
denaturing mix of 1.0M NaoH +1mM EDTA.Incubate at 37 degrees for 30
mins. Then neutralise with sodium acetate pH4.8 ( 1/10 the total volume
of the reaction) mix .Then precipitate with ethanol on dry ice  ,wash
with 70%, dry and resuspend in the required vol.

so usually working from a miniprep it would be

40ul mimiprep
10 ul x5 denaturant
5ul sodium acetate

this would give enough for about 4-6 reactions depending on the
conc.Hope this helps.

Jim hughes



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