What happens to my ligation system? - Very Very urgent! (May/25/2006 )

Hi everybody:

Some weeks ago I amplified a fragment with DyNAzyme EXT DNA polymerase,PCR product Has XhoI SITE ON ethier end. On the 5'endthe XhoI site I add 4 anchor nucleotides,
Anchor nucleotides on the Forward primer is 5'ATCT, while on the Reverse primer is 5'TTCg, I want to digest it directly and the Insert it into the pET, I put the liagtion system in 4C Overnight.
when I pich the colony to identify the recombinat plasmid , I get all religated pET! Can anyone tell me what happens to my ligation System?it really kills me!

Many thanks!

As xhoI is on either side of the PCR, u r trying to ligate this insert with a single digest in your plasmid. U have to dephosphorylate the vector and then try to ligate it or the vector will religate.

What scolix said is right,

Follow these steps
1. Cut the vector with XhoI, dephosphrylate for 15-20 mins (In my experience, more than this has led me to the issues, more harm by phosphatase then help)
2. Cut the gel extracted PCR product. (I prefer gel extracted as it is 'clean', I know few members of my own lab who digest PCR mix directly)
3. Setup ligation overnight at 16-20C (I have never seen any need of 4C unless you are doing it over the weekend and not stopping by the lab)
4. Setup a negative control at the time of digestion, if you are suspicious, that will save your time of checking false positive colonies.

Hi, scolix, Jiang M:

Thanks for your Reply!

I wander whether the anchor nuceotide added on the 5'end of the XhoI sites can not be binded efectively by the XhoI restriction Enzyme? I digest the PCR product with 2 times Enzyme for 3Hr! I wonder whether the Enzyme Cut it completely?