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Transient Transfection - Low Transfection Efficiency (Feb/15/2002 )

I have a problem with transient transfection of Rat-2 fibroblast cells.
I intend to assay the luciferase-reporter gene activity.
The beta-gal is the internal control to check the transfection efficiency.
Every time I get inconsistent results although I use same reagents, same DNA .
What could be the problem?
Secondly, when I do luciferase assay, the value drastically changes between two samples with similar treatment...more funny is that the same sample, when taken reading at different time, the value changes.
By different time, I mean within 5 minutes.
What could be the problem?
Waiting for your reply.


Inconsistency of results of the tests means that the substrate you are putting into the tests is inconsistent, whether you realize it or not ie: you have a quality control problem.  There are many factors which may be varying.  I suggest you list all the aspects of the experiment (eg: cell plating density, time to transfection, size of samples for luminometer etc and define how you measure each.  You will find sources of variation.  I have found the to be a good source of assistance.  Time spent analyzing all these little problems is better than time spent repeating mistakes

-Peter Terry-