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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:

EcoRI-AhdI------2kb Junk-----AhdI-BamHI

Insert via digestion with Eco and Bam into your plasmid. to get:

Plasmid---------EcoRI--AhdI----2kb junk----AhdI--BamHI---Plasmid

When you do a digest with AhdI you get

Plasmid----EcoRI--GACNNT NNGTC-BamHI-Plasmid
Plasmid----EcoRI--CTGNN TNNCAG-BamHI-Plasmid

Note however that a) This has already been done by people before so you can't patent this or anything, and cool.gif I believe promega has a patent on this type of method to make T plasmids.

QUOTE (atefeh @ Oct 27 2005, 04:54 AM)
I want to make T/A vector , anybody can help me ??


thank you very much!
could you tell me what is ingredient of ligating buffer?