Help with Ligation - (Jun/24/2007 )

Hi... I am trying to clone a 5Kb fragment into pBLUESCRIPT by blunt end cloning. I dephosphorylated the ECORV cut vector using SAP and phosphorylated the insert( as its a PCR product from a proof reading enzyme) using T4PNK, and ligate it using 1:5 ratio and I have also tried various ratios. I donot get any white colonies while performing a blue white screen. I also don't know if my controls are working. Am I supposed to see blue colonies when I transform with linear dephosphorylated vector only? or the blue colonies are due to non dephosphorylated vector?? Please help !! I am desperate!!

Firstly I must say that the current strategy is difficult. Blunt end cloning in general is not easily, and blunt end cloning of a large insert is even more so. I would strong urge you to reconsider this strategy. It is possible place restriction sites on primers, allowing the PCR insert to be digest, made to have sticky ends and ligated to a vector with similar sticky ends.

Alternatively you may purchase the TOPOzero (blunt end) ligation kits. These kits have topoisomerase and sscB gene to help ligate and decrease the background of empty vectors.

Dephosphorylation prevents a vector from recircularising. An empty vector is blue as it has a functional lacZ gene. Thus all the blue colonies that you see are either vector molecules that have never been cut or molecules that have not been dephosphorylated and were religated when exposed to the ligation reaction.

However this is biology, nothing is every perfect or clean. And it takes only a single molecule to make one colonies and there is a whole lot of molecules in a ng.

Not any topo cloning. topozero cloning. Regular topo cloning requires A tailing of the PCR product, which proof reading polymerase do not do. Proof reading polymerases only produce blunt end PCR poducts. So once the blunt end product is formed, A tailing using Taq and dATP is required.

As for the restriction digest strategy, did you add enough bp to skirt the restriction site? ie for a restriction enzyme to cut, the restriction site has to surrounded by several bp. The exact number depends on the restriction enzyme.

Since you are already dephosphorlating the vector with SAP, and phosphorylating the insert (with PNK - i assume), the only thing I can suggest is to use more insert, increasing the insert to vector ratio. And using quick ligase. Quick ligase has 15% PEG 6000 (final concentration) and it help crowd the molecules together, giving better ligation efficincy.

Overnight ligation at 16 degress helps. Dropping the temperature to 4 degrees sometimes works.