Ligation help ! - blunt end ligation probs. (Oct/24/2005 )

Hello, M trying to put a PCR amplified fragment into pTZ57R.
1st i digest pTZ with PstI, polish the ends with Moong Bean Nuclease.
2nd i PCR amplify my insert, polish its ends with Moong Bean Nuclease.
3rd I set up an overnight blunt end ligation with T4 DNA ligase.

The problem: I am getting clone with the insert in opposite orientation only. Therotically, there should be a 50-50 chance of the insert in both directions.
The insert is around 1.9 Kb. vector: 2.8.

have purified after all digetsions, so no residual enzymes around.

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Why? Why?! Why not PCR amplify with primers containing Pst I restriction sites instead of doing this blunt cutting stuff? Can't you do a double digest of some sort to prevent orientation problems?

Don't do blunt-end cloning if you can avoid it.

-Matt

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O.K., I just looked at your vector.

Here is how I would do it.

PCR amplify with following primers

GGGCCC[GGATCC]5' sequence

GGGCCC[CTGCAG] 3' sequence (inverse complement)

clone into t-vector/blunt topo vector...or proceed immediately to restriction digest of PCR product.

cut recipient vector with PstI/BamHI in BamHI buffer (NEB U Buffer)

cut insert with PstI/BamHI in BamHI buffer (NEB U Buffer) (preferably overnight)

Ligate with T4 DNA Ligase.

Transform with negative no ligase but cut control and varying concentration of insert to recipient vector.

-Matt

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thanks a lot man, gwtting primers is laast option, back in india, everythigns expensive. i wud require thios just once,...... so....will try some other startegy, last option is primers...will try linkers though....have a few of them.....thanks again.

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I agree, I'd avoid blunt-end cloning if you can. But, it seems from your prior answer that it's not feasible...

But, you are getting clones, they're just all in the opposite orientation, right? Any chance your clone, if in the correct orientation, is lethal to the recipients?

You might also consider cloning your PCR product (using your existing primers) into an intermediate TA vector -- your product would then be flanked by numerous restriction sites, and you could pop it out with sticky ends...

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