cloning problem (pGL2basic/pGL3basic)

Heinz-Juergen Schaefers h-j.schaefers at popHost.com
Mon Nov 11 20:17:17 EST 1996


Hi all!

I have the following problem:

To investigate promoter activity using luciferase assay,
I tried to get a 664 bp promoter fragment into either 
pGL2basic or pGL3basic (PROMEGA) luciferase reporter plasmid.
Using different restriction enzymes for cutting plasmid and
fragment, different digest purification technics (glass milk,
spin coloumns), different ligases/ligation technics, different
transformation technics/competent cells (DH5alpha, JM109, 
chemical- or electro-competent), I was unable to get any
transfectants.
Even transforming of only the religated plasmids did not lead
to any colony.
The only transfectants I got by transforming the untreated
plasmids.

Any suggestions??

-- 
Dipl. Biol. Heinz-Juergen Schaefers
University of Erlangen, Med. IV, Nephrology Research Lab
Loschgestr. 8, D-91054 Erlangen, Germany   +49 (0)9131 85-
E-mail: h-j.schaefers at popHost.com      -9206 (voice) -9202 (fax)



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