In article <firstname.lastname@example.org>,
jlight at RESUNIX.RI.SICKKIDS.ON.CA wrote:
> 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.
Form I DNA probably won't completely separate stands, they are wound
around each other. If you want to separate the strands completely, you
most likely need to cut the DNA. But partial denaturation and annealing
should work. After all, we can sequence dsDNA.
Stephen R. Lasky Ph.D. Brown U/Roger Williams Medical Center, Providence, RI.
Phone: 401-456-5672 Fax: 401-456-6569 e:mail: Stephen_Lasky at brown.edu
America may be unique in being a country which has leapt from barbarism to decadence without touching civilization. John O'Hara.