TA Cloning - (Nov/29/2005 )
I need to convert a particular vector into TA vector and it does no have site for blunt end restriction enzymes. I want to cut with an enzyme that leaves sticky ends, and then converts the sticky ends to blunt ends. Does any one has experience with this, or any suggestion? Thanks.
Why not just get a commercial one? Easiest if you don't would be to add Klenow with DNTPto your digested vector. This will trim back 3' overhangs and fill in the 5'overhangs. This leaves you with a blunt end vector. After that, do whatever it is you were going to do to the blunt vector. However it may be a good idea to heat inactivate the Klenow before adding the T otherwise you would be left with a blunt end vector again. I have blunted vectors like this before when I needed to ligate a blunt ended insert into an EcoR I site.
How big is the vector? Could you amplify the vector by PCR. If you use a proofreading polymerase like PWO (Roche), then you will be left with blunt ends at the 5' end of the primers. Your PCR product/vector will also be dephosphorylated..extra bonus! Of course, if you want them to be phosporylated you could order primers with incorporated phosphates or add the phosphates enzymatically afterwards.
I also gave another guy a description of how to make one that can be easily reproduced which can be found here
Thanks! Your suggestion fits into my idea on how to go about it.