Variability Between Plasmid Preps in Transfections

Brian Foley brianf at med.uvm.edu
Wed Aug 3 13:39:32 EST 1994


	In my experience the only way to get truly quantitative results
between plasmids and transfections into mammalian cells is to use an 
internal control.  I use two markers on the same plasmid, one is my
gene of interest, and the other is Hygromycin resistance, CAT, or
some other quantifiable reporter.
	Are you sure that all of your plasmid preps have equal ratios
of supercoiled:relaxed forms of plasmid?  Run an equal mass of each 
plasmid on agarose and have a look.
	CsCl prepes should give good transfections, but some people have
reported that Qiagen columns produce DNA that gives more transformants
per microgram of plasmid.
	CaPO4 precipitation is OK, but it might be easier to standardize
transfections with Lipofectamine or Transfectam.  I find that Lipofectamine
is great for transient expression, Transfectam is better for stable
transfections.  
	
	I have seen many publications in which the prper controls are
not reported.  They just transfect with two plasmids and report any
differences as differences in promoter activity, ignoring the possibility
that the differences might be due to unequal transfection eficiencies.  


 --
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*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *
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