Creating T-cloning vector

Mike Prigge prigge at darkwing.uoregon.edu
Mon Feb 22 20:49:21 EST 1999


In article <7as1pv$gjr$2 at unix2.glink.net.hk>, "Andrew Leung"
<leungkc at glink.net.hk> wrote:

> In Duncan's response, I just wonder would ddTTP be compatible for ligation.
> I mean ligase can join 5'-nnnnn ddNTP to 3'-dNTP nnnnn in DNA nick?
> 
> Andrew
> 

Unless you phosphorylate your primers, that 3' wouldn't have anything to
be ligated to anyway.  It is only the 3'-OH on the overhanging A of the
PCR product that need be involved.  E. coli takes care of the rest after
transforming.

Start:

--T/A--NNNGATT-H     H-NNN---PCR---NNNA-OH  PO4-ATCNNN--T/A--
Vector-NNNCTA-PO4  HO-ANNN-Product-NNN-H     H-TTAGNNN-Vector

Ligation:
             H H                                                          
             | |                                                           
--T/A--NNNGATT NNN---PCR---NNNA-ATCNNN--T/A--
Vector-NNNCTA-ANNN-Product-NNN TTAGNNN-Vector
                             | |
                             H H

After E. coli Replication:

--T/A--NNNGATT-NNN---PCR---NNNA-ATCNNN--T/A--
Vector-NNNCTA-ANNN-Product-NNN-TTAGNNN-Vector

I hope this ansers your question.
Mike
-- 
Michael Prigge                          Phone: (541) 346-4256
Institute of Molecular Biology            Fax: (541) 346-5891
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