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Transient Transfection

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#1 Gunasekaran



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Posted 15 February 2002 - 01:14 AM

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.

#2 Peter Terry

Peter Terry


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Posted 26 February 2002 - 06:54 AM

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 www.lifetech.com to be a good source of assistance.  Time spent analyzing all these little problems is better than time spent repeating mistakes

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