Self ligation of vector - (May/19/2005 )

Hi
I`m trying to clone an insert into a vector (pcDNA3). I have performed the digest cut with 2 enzymes (SacII and KpnI) in both vector and insert. It seem the digest cut was partial but I did obtain a couple of bands (insert and vector) of the right sizes. I performed a ligation reactionwith vector and insert and with vector only. Unfortunately, I have a lot of colonies in the vector only dish, much more than in the dish with the same amount of vector than in the vector only dish together with insert. Is there any possibility that the less efficient growth of colonies in insert plus vector dish is due to the fact that they did ligate or do you think it is only vector self-ligation?

Hi!

Why don't you try to desphosphorilate your vector with SAP(Shrimp Alkaline Pshosphatase, from Roche)?? Then you can have a negative control of the ligation (vector without insert). Maybe your problem is that the vector religate, but if the vector is desphosphorilated the % of religated vector is less.
good luck!

Hi trosenba,

If your vector digest is incomplete then it is like trying to find a needle in a haystack to find the insert, especially without blue/white screening.

One trick to remove the undigested vector from the digested is after the ligation digest it with a enzyme with a recognition site within the area that is excised and thereby linearise any undigested vector and then transform it.

It is unlikely that your your insert is effecting the growth of the cells as the protein won't be expressed in bacteria with a prokaryotic promoter to drive transcription.

Hope this helps

Scott