How to make a T/A vector - (Oct/27/2005 )
I want to make T/A vector , anybody can help me ??
You can buy such vectors from promega. If you want to make one the easiest way to go is to use the Ahd I restriction enzyme. Engineer the Ahd I site in two primers (a forward and a reverse) and PCR a large enough fragment of DNA (why a large one? It will be obvious in a second). Since the Ahd I site is GACNN_N'NNGTC, all you have to do is ensure that the overhanging end is a "T". Insert your PCR'd fragment into a vector of your choice and transform. Now when you want a linear T vector simply take your plasmid prep, digest with Ahd I and gel purify the digested plasmid. Since the insert that is removed is large (1-2kb) you should have no problem telling the digested vector from undigested. So ex/
Primer Fwd: EcoRI-AhdI---->
Primer Rev: BamHI-AdhI--->
Do PCR and get this band:
Insert via digestion with Eco and Bam into your plasmid. to get:
When you do a digest with AhdI you get
Note however that a) This has already been done by people before so you can't patent this or anything, and I believe promega has a patent on this type of method to make T plasmids.
thank you very much!
could you tell me what is ingredient of ligating buffer?