Beta-gal cotransfection - nonsense results?

Jorge L. Sepulveda Jorges at bcm.tmc.edu
Fri Dec 4 11:54:16 EST 1992


There are two papers that may be relevant to this discussion: 

 1) Farr, A and Roman, A, Nucleic Acids Res., 20, 920, 1992; the authors 
showed interaction between a test promoter linked to CAT and RSV-b-gal; a 
possible solution is 

2) Abken, H and Reifenrath, B, Nucleic Acids Res., 20, 3527, 1992; CAT 
activity is normalized by doing DNA dot blot analysis of the amount 
transfected plasmid (no co-transfections!). My problem is that the authors 
mention that they lyse the cells (HOW?), do CAT assay on the crude 
lysates and dot blot after treating with RNAse and Proteinase K but don't 
give enough details to reproduce the experiment.

Is there anyone who might have a good protocol for assaying the amount of 
transfected plasmid by hybridization? Wouldn't it be preferable to do it 
in the same lysate used for CAT, both for simplicity and minimization of 
variability?


Jorge L. Sepulveda, 

Dept. of Pathology,                        Email: Jorges at bcm.tmc.edu
Baylor College of Medicine                 Telephone: 713-798-7330
One Baylor Plaza, Houston, TX 77030        Telefax: 713-798-5838



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