PCR A/T CLONING VECTOR

[Michael Cooley]

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!!!