PCR A/T CLONING VECTOR

Richard T. Timmer rttimme at unix.cc.emory.edu
Tue Dec 20 18:24:36 EST 1994


Michael Cooley (szcooley at dale.ucdavis.edu) wrote:
: marcvdc at lmb1.rug.ac.be wrote:
: : IS  IT POSSIBLE TO MAKE A PLASMID VECTOR WHICH GENERATES  SINGLE T-ENDS AFTER 
: : DIGESTION WITH A CERTAIN  RESTRICTION ENZYME ? 

: : Such a plasmid would be very usefull for cloning PCR-fragments by  A/T 
: : cloning.  The advantage would be that you only need to digest the vector to be 
: : certain that  all the 3' ends contain only one T .


: : OR DOES ANYONE KNOW WHERE I CAN BUY SUCH A PLASMID ?

: : I realise that clonetech and promega both offer PCR cloning kits but they only 
: : offer linearised plasmids generated by ddTTP tailing of blunt end plasmids.



: : Thank you in advance for your nice reply

: : MARC  VAN DE CRAEN

: : E-MAIL:  MARCVDC at LMB1.RUG.AC.BE



: I was sent a protocol for making a T/A cloning vector using the TAQ 
: polymerase to add the necessary T's to the end of a blunt cut vector. I 
: haven't tried it as yet, but considering the ridiculous cost of 
: Invitrogen's plasmid I am likely to try it soon. YOU HEAR THAT INVITROGEN!!!


Could you post your protocol for generating the T/A cloning vector using 
TAQ?  Thanks. 

Richard Timmer
Dept. of Physiology
Emory University
Atlanta GA 30322-3110



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