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Troubles with cloning - No positive ligation (Oct/04/2009 )

Hi,

I have for the past week been attempting to clone several things into two different pET vectors (pET17b and pET22b).
I am able to get suitable quantities of PCR product following agarose gel purification (>150ng/uL) and also have no trouble performing double digests of both vectors as determined by agarose gel (using SYBR safe as a DNA stain).
I am using the enzymes NdeI and XhoI (NEB). I have also designed the primers such that I have a suitable flanking sequence 5' of both restriction sites for my PCR product (therefore I would assume that they digest).
Following digestion I Alkaline phosphate (NEB) treat the vector followed by heat inactivation at 65C for 5 minutes gel purifcation, while I simply EtOH precipitate my digested PCR product.

I have tried both 1 hour and 16 hour ligations at 16C with no success.
I have also tried 3:1 ratios of insert:vector or otherwise a crapload of insert to vector...nothing

Negative control - digested vector and no insert yielded no colonies
Positive control - digested vector without phosphatase cleanup or removal of insert produced colonies

Any suggestions would be extemely helpful

Thanks

Paddy

-PaddyS-

Some things to think about:
* Your CIP treatment may be trashing the ends of your vector. Try without it or use minimal amounts or switch to antarctic phosphatase. How are you inactivating the CIP?
* How many bases of overhang 5' of your restriction sites are on your PCR primers? Check that they are long enough.
* How are you purifying the PCR product prior to digestion. This is critical to avoid filling in of the overhangs.
* I sense that you think more DNA is better. This is not true, and you should do ligations with limited amounts of DNA, something like 20 ng of vector and equimolar amounts of insert in a ligation reaction.
* Test the competence of your cells. You will need around 10^8 cfu/ug of plasmid. A few positive control with boatloads of plasmid DNA does not test this.

-phage434-

Hi

Thanks for your reply
Sorry I wrote it wrong, I use Antarctic phosphatase (not Alkaline) and I heat inactivate this according to the instructions (5 minutes)

I will attempt the ligation with those concentrations of DNA today :) Thanks for you help

Pat

-PaddyS-

I quote all the suggestion of the previous user.
I want just to evidenciate that in a "directional" cloning as you are trying to perform, treatement with phosphatase is, not only unnecessary, but according to many investigators (Molecular cloning 3 Ed. Sambrook and Russell), could be negatively affecting the results of cloning...

Therfore I could suggest to use phosphorylated plasmid instead....

Good luck

-Alinor19-