Heat denaturation of Sequencing

daj (David Johnston) daj at nhm.ic.ac.uk
Wed May 4 03:00:25 EST 1994


On 4 May 1994 06:11:33 GMT,
  Dan Diaz writes:

>
>i've had the best success using USB's quick denaturation plasmid sequencing
>kit.  you add NaOH to your plasmid-primer mixture and incubate 10 min at
>37 'C.  then add HCl, reaction buffer and anneal and go.

I've not tried the commercial version of this. With a homemade one I got 
variable results. Out of a batch of say 15 reaction sets (multiple 
templates, multiple primers) approx half would work stunningly (better than 
std alkaline denaturation) and oh so quick, the other half failed 
completely, absolutely nothing on the gel. There was no logical 
consistency to the successes/failures, one template would work with one 
primer and not another (but would work with it using standard 
alk den) and one primer would work with one template but not another (but 
again would work with it using std alk den). I used the same pipetter and 
didn't change its setting between the NaOH and HCl.

Its potentially a marvellous timesaver, I would be interested to hear if 
the commercial kit has the esame problems or is more "reliable".

Cheers
DAJ

David A. Johnston
Dept of Zoology, The Natural History Museum, Cromwell Road,
South Kensington, London SW7 5DB. England
(tel 071 9389297, fax 071 9388754, email daj at nhm.ic.ac.uk)



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