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Self-Ligation of digested vector


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#1 Signal

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Posted 20 June 2009 - 03:57 AM

Hello/Nimen Hao

I got pCHF3 vector from someone but it is digested with BamH1/Sal1. What I want to do now is to self-ligate the vector to clone a gene using different enzyme sites. What should I do, I know! but I need some suggestion because I might be wrong.

1-Let the vector self-ligate in the presence of T4-DNA ligase or some other ligation solution, how much should be the reaction volume? say for example
Vector 2.0uL+3uL Ligation solution?
2-What verification step I can do after E-coli transformation to confirm that the vector is successfully self-ligated? Maybe I should run both digested and self-ligated vectors to see the difference?

Any suggestion will be highly appreciated ;) ...

Edited by Signal, 20 June 2009 - 04:10 AM.

I/we have to wait for 30 years to let the mirAcle haPPen....someone here told me!!!!

#2 Signal

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Posted 20 June 2009 - 07:47 AM

Well, I think its impossible to get the vector in its original form after self-ligation because the vector has already lost its MCS after double digest. ;) ....
I/we have to wait for 30 years to let the mirAcle haPPen....someone here told me!!!!

#3 pcrman

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Posted 20 June 2009 - 07:52 AM

Hi Nihao,

Have you taken a look at this topic on how to create an empty vector?
http://www.protocol-...?showtopic=7379

Yes the MCS sites within the two RE are gone. You have to sequence the plasmid to determine the exact sequence at the ligation site.

If you get colonies after self-ligation and transformation, it tells you that the ligation occurred because only circular plasmids can transform the bacteria and are antibiotics resistant.




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