pGEM-T cloning

user unknown ez005679 at dale.ucdavis.edu
Sat Jul 13 12:58:50 EST 1996


Christoph Grunau (cgrunau at imb-jena.de) wrote:
: In article <Pine.SOL.3.91.960705133209.533B-100000 at tin>, "Miss. V.M.
: Hodges" <vhodges at hgmp.mrc.ac.uk> wrote:
: 
: > I've recently switched from using InVitrogen's TA kit to the pGEM-T 
: > vector from Promega to get both the sp6 and T7 promoters in one vector.
: > I've found that in my hands the cloning efficiency of the pGEM vector is 
: > really low despite having tried a number of vector:insert ratios.
: > Has anyone any suggestions?
: > The transformation efficiency was good but  very few colonies contained 
: > inserts. I have been using blue-white selection but I'm 
: > getting very few colonies on my test plates to start off with and most of 
: > the colonies were blue . Even clonies that were white had small blue 
: > centres but still had no insert.
: > 
: > all replies gratefully recieved
: > Vivien
: 
: Did you try the control insert they supply with the kit? If it doesn't
: work neither send it back to Promega and ask for a new one. 
: 
: However, in our hands the cloning efficiency was always a bit lower for
: pGEM-T than for the TA-kit from Invitrogene. But the pGEM-T kit is much
: cheaper and we usually get absolutely enough inserts (efficiency 96 -
: 25%). You can reduce the recommended amount of vector to 12.5 ng in 10ul
: and it still works fine. 
: I precipitate the ON ligation reaction and do electroporation.

Agree.   pGEM in my case is about 1/2 efficient as TA cloning, but very 
cheap.   Purification of insert before cloning might be important.




: 
: Good luck - Christoph
: 
: -------------------------------------------------------------
: Christoph Grunau
: AG Genomanalyse
: Institut fuer Molekulare Biotechnologie e.V.
: Beutenbergstr.11
: D-07745 Jena
: Germany
: 
: Tel.: +49 (03641) 65 62 42
: Fax.: +49 (03641) 65 62 55



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