pGEM-T woes

Gerard T O'Neill gpva11 at udcf.gla.ac.uk
Wed Dec 14 04:30:26 EST 1994


re: pGEM T vectors:

Ihave used T-vector from Promega on several ocassions.
I do ligations at 15 C for ^18h.
I have had few problems and on sequencing, usually find that the only
modification to the MCS is the T insert.
Nowever I know of others who have had bad experiences.
I only select the white colonies; my only grudge is that the white/blue
ratio is low.
gerry oneill
> 
> Rae Nishi (nishir at ohsu.edu) wrote:
> ...
> : batch we've gotten has had problems.  For example, one lot had an
> : unacceptably high background of white colonies when the plasmid alone
> : was used to transform.  Another lot seemed to be missing the T-
> : overhang so that we couldn't get even the control insert to work.  More
> : recently, we've had a more insidious problem... two clones (with
> : different inserts, each prep done by a different person in the lab)
> : that we isolated are missing the T7 end of the vector; that is, none of
> : the enzymes that are in the multiple cloning site on the T7 end of
> : where the insert should go will cut.  We (now) know all our enzymes are
> ...
> 
> I've used pGEM-T with generally acceptable results, but I use a 
> ligation time of about 24 hr.
> However, I noticed in some of the minipreps that the isolated plasmids
> had major deletions somewhere near their MCSs. This concerned only the 
> white colonies and never the faintly blue ones which were usually correct
> ones (in some cases, the white ones also were correct). 
> The proportion of deletion-containing plasmids seems to vary between 
> lots, and right now I have a good kit in my hands (not literally).
> 
> Anyhow, Promega's products are much more reliable than Clontech's:
> I used the PCR MIMIC Construction Kit, and it had a significant deletion
> in the template DNA. After complaining, they sent me another tube.....
> it had the same deletion. 
> 
> TH
> 
> 
> 




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