To dephosphorylate the vector or not? - For cloning PCR product (Jun/03/2006 )

Hi everybody!
To dephosphorylate the vector or not? I try to clone the PCR product into plasmid both cut with two restriction enzymes and get a lot of colonies. As I know phosphorylation after PCR is insufficient, so dephosphorylating the plasmid I prevent the ligation. Am I right? Thanks.
Sem

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What I feel is, if vector is cut with two enzymes then dephosphorylating is not very necessary but still its better to do because, after double digestion both the enzymes might not have cut and you may have linearized vectors with two different ends and some with same end! SO in control you may get colonies after ligation.

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I agree with Calvin.

As long as u know the enzymes u have used cut efficiently then u dont need to dephosphorylate. Ofcourse dephosphorylation is recommended for blunt end ligation. If u digested ur vector completely, then u shouldnt get any background colonies.

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Hi, Calvin and scolix!
Thank you for your reply. But look, if you dephosphorylate the plasmid plus the fact that PCR products are usually insufficiently phosphorylated- theoretically you are not suppose to get ligation!!!
Thanks.
sem

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hi
you pcr product is generally digested after pcr
so you have phosphorylated 5' basically (that's wh we dephosphorylate vectors...)
so pb does not came from your pcr product, except is you've blunt ends (and maybe the 3 phosphates are not good for ligase.

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Hi

Sorry, I dont understand you: you mean p base does not came from the vector, but from the digested PCR product? (because we dephosphor. the vector and digested PCR product has P on him?)
Thanks ,
sem

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that's right.
when u cut your pcr product, u get ends with phosphates. so when u dephosphorylate your vector, ligation will proceed with the phosphates on your pcr product.

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