T/A cloning

Mon Jan 8 12:44:08 EST 1996

On 8 Jan 1996, Helen Korsmo wrote:

> We ligated an insert into a T-vector (after treating the insert for 30 minutes with Taq; in order to 
> put A's on the ends) and transformed DH5-alpha cells and plated out on LB plate with IPTG and 
> X-gal.  We got several white colonies ( presumably these have insert in the vector), but mostly 
> blue (vector alone).  Is it possible for T-vectors to ligate?
I see the same thing while using pGEMT vector. I think due to the single 
base overhang (and because this vector is not dephosphorylated) the vector 
essentially behaves blunt ended and undergoes self ligation. The problem 
(if it is one) is overcome by using an excess of insert DNA. Still, there 
will be some blue colonies. 

			  Hiranya S. Roychowdhury
   			  Plant Genetic Engineering Lab.
			  Box 3GL, NM State Univ.
			  Las Cruces, NM 88003
			  Phone: (505) 646-5785
			  hroychow at nmsu.edu

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