Beta-gal cotransfection - nonsense results?

Michael Holloway ca566 at cleveland.Freenet.Edu
Wed Nov 25 19:29:21 EST 1992


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

>[description of co-transfection results deleted]
>
>Well, the CAT results are not *completely* turned around in
>Biotechniques (13:700) but some of the relationships change.

Granted, I am making an assumption that the list of CAT constructs they
have are a nested deletion set listed in order of length.  We won't know
untill the JBC paper appears, but I'll bet money that I'm right.  If the
assumption is true, the clear progression of the CAT results IS turned
around completely.  The progression of the results is completely reversed
based on Bgal cotransfection data that varies 10 fold.  The Bgal data
varies progressively, from the top of the list to the bottom.  This
progressive change in Bgal makes no sense to me and makes the use of it
highly suspect.

>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.  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?

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.

Thanks
Mike

-- 
E-mail: mhollowa at ccmail.sunysb.edu (mail to freenet is forwarded)
phone:  (516)444-3612



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