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strange results when cloning long PCR fragment
Posted by: Christof (IP Hidden, New member, 1)
Date: July 13, 2006 06:45AM

Hi,

I`ve got some problems when cloning a 5kb PCR fragment.

For the PCR Im using Accu prime polymerase (Taq+proofreading enzyme). On the agarose I can see a single band at 5kb - so the PCR seems to work.
After purification I`m putting on a pure Taq polymerase at 72C for 30min to get A-overhangs. The cloning vector is pDRIVE (3.8kb) from Qiagen with U-overhangs. After ligation at 4C for 24h and transformation in electrocomp ecolis I get blue and white colonies.
As expected the blue colonies are religated vectors (3.8kb) when cutting with restricion enzyms and size confirmatoin by electrophoresis.

But..when checking the white colonies I always get a singe band at 5kb (but should be 9.8kb) after digest with a single cutting enzyme.

Befor using the PCR cloning vectors with U-overhangs I did bluntend ligations.
There the same happend: I got bands of religated vector (2.3kb) and bands at 3.5kb.

It would be great if anyone could help me with this problem - I`m getting a bit desparte!

Christof

 

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