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Directional cloning

Directional cloning - Protocols and Methods Forum

Directional cloning - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 03-27-2006, 03:33 PM
Jayakumar, R
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Default Directional cloning





If the cut vector has incompatible ends, de-phosphorylation is not
necessary. I think you may be having a digestion problem rather than
religation problems (ie., if you are using double digested inserts and
vectors). Did you check the ligated vector mix on a gel to check for
religation. What about control transformations with control ligations
(no insert) and unligated vectors (undigested)? They should help you to
pinpoint the problem.

Best of luck
Jai



-----Original Message-----
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[mailto:[Only registered users see links. ].indiana.edu] On Behalf Of newsnet
customer
Sent: Monday, March 27, 2006 3:31 AM
To: [Only registered users see links. ]
Subject: Directional cloning

Hi,

I performed directional cloning of a gene into a vector.

I was wondering if you need to de-phoshorylate the ends even with
directional cloning where the cut vector does not have compatible ends?

It would seem possible that a cut vector could ligate with another cut
vector (not self ligate), so it would be a good idea to de-phoshorylate
the ends when doing directional cloning?

What are peoples opinion? I just screen about 10 colonies and they are
all negative, so there must be some form of re-ligation happening.

Cheers,
Sharp
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