I'm doing a subcloning experiment with a pTNT vector and I treated it and my inserts (engineered with RE recognition sites) with EcoRI and KpnI, then incubated the digested vector with alkaline phophatase. I did a control experiment with each enzyme individually to confirm that they were both active. Upon doing a ligation experiment and screening the colonies, all of them had circular vector with no insert. To make matters more confusing, I was able to successfully ligate one gene into the double digested pTNT vector already, so I'm completely baffled as to why my vector is now showing signs of self-ligation.
Also should clarify: I am using calf intestine alkaline phosphatase, inactivating after digest by incubating at 65 deg C for 20 mins.
There is no such thing as a reaction going to 100% completion. The transformants yielding recircularized vector arise from vector molecules that were incompletely digested by one or both of the restriction enzymes and further escaped dephosphorylation by akaline phosphatase. Recircularization is a very efficient reaction, thus even a minute fraction of the vector that is cut only once and not dephosporylated will be recircularized and and show up in transformed cells. In order to avoid such background, you can treat the ligation mixture before transformation with a restriction enzyme whose site is located between the two restriction sites of the vector that you chose for cloning and that is of course absent from the cloned insert
Thank you for the response Pierre. Regarding background, I also do a gel extraction on my Endonuclease/phosphatase treated vector to further isolate the cut vector from the remaining uncut vector, is that insufficient to reduce the background that you described?
- Badges
- Science topic
- Similar topics
- Vectorization