CAT assays-reproducability

Fri May 28 05:12:00 EST 1993

Dear All,
         Are there any old fashioned CAT assayers out there?
     I am engaged on a project in which I am attempting to analyse various
putative enhancer elements for response to oncogenes (eg src, int, hst etc).
We are currently using a reporter system based on the HSVtk promoter ie 
pBLCAT2. The big problem has been reproducability of the response. I seem to be
able to get everthing from 30-fold induction down to nothing from experiment
to experiment. Is this level of variation unusual? If so, any ideas on
minimising it? 
  Another problem is that the negative control ie pBLCAT2 containing no added
enhancer, appears to respond up to 10x to oncogene transactivation, minimising
any putative enhancer effects seen on the elements under test. 
  Several other groups use this reporter system with no such apparent problems.
  We have tried controlling many perceived sources of variation with little
lasting success, I can go into more detail with any respondents.

Graham Atherton

More information about the Methods mailing list