Why my construct cannot be cut? - (Feb/01/2001 )

I subcloned a PCR fragment to a vector. Sequencing showed the the fragment was successfully inserted to the vector, but I could not cut the insert off using the two enzymes for cloning. Who can tell me why?

If you are using both enzymes in the same reaction, make sure that they are compatible as far as buffer conc. and temperature goes. Also be sure that the glycerol does not compose more than 5% of the reaction. (Enzymes are usually in 50% glycerol. Thus you can only use 2.5ul of each enzyme in a 50ul double digest total reaction, for example.) I would suggest doing the following diagnostic: Start with clean plasmid and set up two separate reactions, one for each of the two enzymes you are using. Clean these up after the digests by P/C extraction and run them on a gel along with some uncut plasmid. The linearized plasmid should run a little slower than the uncut plasmid. Next, take each of the two clean linearized plasmids and use the corresponding, second enzyme for each reaction to give a double digested product. Clean this up and run them on a gel. Hopefully, if it is a problem with enzyme compatibility, this should shed some light on things.

Good luck!

Can you say it clear. Which restiction site you use? Ben-Tsu