Beta-gal cotransfection - nonsense results?

Stephen Klautky agoodrid at vaxa.weeg.uiowa.edu
Thu Nov 26 00:10:00 EST 1992



In article <1f15p1INN46q at usenet.INS.CWRU.Edu>
ca566 at cleveland.Freenet.Edu (Michael Holloway) writes:

>In a previous article,
>agoodrid at vaxa.weeg.uiowa.edu (Stephen Klautky) says:

>>I (and probably others) would like more information about your
>>transfection protocol.

>>Is the total amount of plasmid (micrograms) the same for each transfection?

>>Is the amount of plasmid for the test promoter held constant
>>or is the number of moles held constant?

>>Have you tried different preparations of plasmid with the same
>>result?

>I've got the second attempt cooking now.  It uses a second preparation of
>plasmids.

Well, I don't put too much stock in the results of one transfection.
It's best to do them at least 3 times (though if the data are close
you can get away with 2).  

>    That first time, I used a total of 40ug of plasmid based on a 
>protocol in a neighboring lab for making stably transfected PC12's. 20ug
>of test plasmid and 20ug cotransfected.  The closest thing to an
>explaination I can come up with, or that anyone here has suggested, is that
>this is really too much plasmid or that the RSV enhancer/promoter on the
>Bgal is competing with the test plasmid for some limiting factor.  This
>time I've limited the Bgal to 5ug.

>I've never seen a transfection protcol that payed any attention to the
>number of moles of plasmid even though it does seem obvious that it should. 
>Can you or anyone out there point out some reference where someone has
>looked at this?

I might have a reference or two, I'll have to look.  But in the meantime
I can tell you that that is what I did - transfect equal moles of test
promoter.  I have 5' deletions from 6 kb to 59 bp (to +31); my plasmids
range in size from 11 kb to 5 kb.

I used RSV-luciferase for my transfection control.  However, since then,
others in the lab have begun using CMV-beta-gal.  Someone else replicated
my results with the beta-gal construct (without strange trends in beta-
gal activity) so no problem there.

We try to use the smallest amount of control plasmid that is easily
detectable - just do a titration curve.  Part of the reason is the one
you stated above, that is, potential competition for transcription
factors.  I use a ratio of 10:1 or 5:1 test promoter/control plasmid.

>Thanks for the interest.  For the most part I've just been shocked by what
>I thought might be a common artifact produced by someone like me just 
>starting to do CAT assays.  Bgal results are not commonly reported after 
>all, just the normalized results.

One would hope that obvious trends in the control plasmid's activity
would be dealt with before an attempt to write the paper.  But you never
know.

In some cases you don't need to normalize your test promoter's reporter
activity.  If you can demonstrate that your transfections are consistent
within a given preparation of cells there's no reason to add a another
complicating factor.  I know of two labs that can do this - but only
after a number of transfections to characterize the system.


Earlier you wrote:
>As the size of my upstream region decreased in a nest set of CAT
>reporters, the Bgal numbers went down till there was a factor of
>10 difference between the highest and lowest.

I would have thought that competition would be lessened as you
decrease the amount of 5' flanking sequence; presumabably you remove
cis-acting elements and thereby increase the concentration of
available factors.  But I suppose that assumes that the activity
of the test promoters decrease as you delete sequence 5'.

Steve     AGOODRID at VAXA.WEEG.UIOWA.EDU



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