CAT Assay blues

Soeren Jensby Nielsen sjn at biobase.dk
Fri Jan 19 09:02:43 EST 1996



Hi netters

We are curently encountering a problem of inconsistency in our CAT assays.

We co-transfect HeLa cells with a beta-gal expression vector (as an internal
transfection standard) and a CAT expression vector where the CAT gene is
driven by our promoter of interest.
 We do transfection mastermixes in the sense
that we do every construct in triplicates. Within each triplicate the beta-
gal assay is very consistent but the CAT assays are very jumpy. This is frus-
trating as it defies all commom sense.
The specifics are:
Transfection : CaPO4 mediated coprecipitation
Beta-gal Assay: colorimetric detection using ONPG
CAT-assay: Phase extraction assay described by Seed and Sheen
Extraction: Trypsinization,wash in PBS,lysis addition of 250mM Tris followed
by 5 x freeze/thaw in liquid N2/37 degrees waterbath, heating of extract
for CAT-assay at 65 degrees for 10 min


Any help is GREATLY appreciated

cheers,
Soeren




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