cloning oligos

Zhonglin Chai via methods%40net.bio.net (by Zhonglin.Chai from bakeridi.edu.au)
Thu May 24 16:26:08 EST 2012


You are theoretically right, but you may find that there are many background colonies without inserts since some vector DNA may be cut only once by either of your enzymes. This type of DNA is not separable from double cut molecules and self-relegated with a much higher efficiency than vector/insert ligation. You may find it more efficient to obtain your construct by dephosphorylating your vector and phosphorylating your oligo inserts.

Zhonglin Chai

Sent from my iPad

On 25/05/2012, at 2:31, "Ed Siefker" <ebs15242 from gmail.com> wrote:

> I'm trying to clone a short sequence by annealing two complimentary
> oligos with incompatible overhangs.  Am I correct in understanding
> that if I'm doing directional cloning and don't dephosphorylate my
> vector, I do not need to phosphorylate the oligos?
> 
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