How to denature dsDNA and prevent re-annealing?

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Mon Sep 18 16:33:40 EST 2000


In article <8q5hcq$r02$1 at nnrp1.deja.com>, mbraden23 at my-deja.com writes
>Does anyone know of a simple denaturing of DNA method and reagent
>conditions to prevent re-annealing?

Alkali - simple and works.

Biotechniques vol 18. No. 3, p376 (1995)

Basically:

1.      incubate DNA in 0.4M NaOH, 4mM EDTA for 8 mins at 37C

2.      Neutralise with 0.1volume of 2.5M NaAcetate pH 5.2

3.      Ppt by adding 3 volumes EtOH.

4.      70% ethanol wash the pellet.

Works for plasmids and seems to be OK for linear DNA.

Duncan

Working the night shift!
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk






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