Denaturing ds DNA for sequencing

"Alexander Kraev" bckraev at aeolus.ethz.ch bckraev at wawona
Mon Mar 6 11:13:37 EST 1995


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

It works, but the reason why most people use alkali is that for practical
purposes it tends to give better results ( than heating ) with less than pure
DNA. The likely explanation for this phenomenon is that alkali also hydrolyses
RNA and the 3'-ends thus generated cannot act as primers. With very pure DNA
there should be no difference, however, the faster you do your sequencing after
neutralization and/or cooling, the better. There are also protocols, where one
first precipitates the required amount of DNA, then adds alkali and the primer,
makes an incubation, add annealing buffer premixed with HCl, anneals and goes
on with sequencing. 
*************************************************************************
Alexander Kraev, Ph.D.                 Internet: bckraev at aeolus.ethz.ch
Lab. of Biochemistry III               Phone: 0041-1-632-31-47
Swiss Federal Institute of Technology  FAX:   0041-1-632-12-13
Universitaetstr.16
CH-8092 Zurich
"Some ideas are obscure not because they are complex, but because they 
 are excluded from our circle of comprehension" - Kozma Prutkov



More information about the Methods mailing list