Denaturing ds DNA for sequencing

Mr SMNN Faruque s.faruque at miranda.umds.ac.uk
Sun Mar 5 11:04:44 EST 1995


In article <3j89lb$f3b at masala.cc.uh.edu>, benedik at uh.edu (benedik at uh.edu) wrote:

: Here is a stupid question for which I am sure there is an obvious
: answer that I just don't know.
: 
: When denaturing ds plasmid DNA for sequencing, why do we go to all the
: trouble of adding NaOH, then neutralizing and ETOH precipitating? Why
: doesn't it work just heating the DNA to 85oC then chiling on ice? I am
: sure there is a reason, but I can't think of it.


I've tried a method mentioned in this newsgroup (sorry but I can't
remember who said it) which is:-

Mix template and primer and make up to 8ul with water.  
Heat at 95degC for 5 minutes.
Pulse spin in a microfuge.
Add 2ul of annealing buffer (as in protocol).
Leave to anneal at 37degC for 15 minutes.

While my sequencing is not perfect, I've found this method to work as well
as alkaline denaturation, if not slightly better 'cos it eliminates some
loss of DNA in the precipitation step.

Nadeem



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