Denaturation of plasmid DNA

Ole Skovgaard olesk at RUC.DK
Tue Dec 19 11:09:42 EST 1995


In article <v01510101ace3bb7fd83f@[142.20.8.21]> jlight at RESUNIX.RI.SICKKIDS.ON.CA writes:
>From: jlight at RESUNIX.RI.SICKKIDS.ON.CA
>Subject: Denaturation of plasmid DNA
>Date: 30 Nov 1995 16:52:45 -0800

>Here's a basic question for which I should know the answer.  Basically, I'm
>working with a mixed population of cDNAs cloned into a plasmid and I want
>to anneal a primer (with an affinity tag) to the specific constructs in
>solution to try and recover the homologous constructs intact.  When you
>want to separate the strands of plasmid DNA and you have mostly ccc DNA
>(say, from a Qiagen prep), what's the best way to separate the strands only
>partially so that when you reanneal, one strand from any particular plasmid
>will reanneal with the other strand from the same plasmid molecule, rather
>than those of other homologous plasmid molecules?  Please don't flame me if
>you think this is stupid - I'm trying to be serious.  Thanks in advance for
>your help.

I really do not think that it is possible to get the mixed molecules because 
the ccc DNA means that the two strands cannot swim away from each other. If it 
was linear DNA that would certainly be another story




******************************************
****   Ole Skovgaard                  ****
****   Dept. Life. Sc. & Chemistry    ****
****   Roskilde University, DK        ****
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