DNA prep for transient transfection

Ken Saville saville at umail.umd.edu
Tue Mar 7 09:29:37 EST 1995


>Hi there,

>As a coincidence, we had a discussion only yesterday about problems with low 
>efficiencies of transient transfections in our lab.

>More precisely, transfections using the DEAE-dextrane method seem to give > >very 
 >variable results, given one plasmid preparation to another.

>The low efficiency is independent of the person, the acceptor cells or the > > DNA 
>itself. In fact...the problem seems to be the preparation method used. Sorry 
> to tell you so, but this all began when we started using QIAGEN columns!

>I am not trying to start a "WIZARD (TM)"-like hystery here. We just want to 
> know if other people have experienced similar problems, and if there is any 
> solution to this. Especially if you want to compare two different genes, > >this 
> varying efficiencies really make it a mess.

>I hope someone has an answer to this problem.

        Greetings,

        Nick

>Laboratory of Molecular Biology
> University of Gent
> BELGIUM
> Nick at lmb1.rug.ac.be


Nick,

Although I don't use quiagen prepped DNA for transfections, I have
encountered some strange results that may relate to your problem.  I
have been using CsCl prepped plasmid DNA in transposition assays in
Drosophila embryos.  Briefly, DNA is injected into embryos and the
movement of a genetically marked transposon is monitored by following
the fate of the markers in E. coli. That is, DNA is re-isolated and
transformed into e.coli.  For example, insertions into lacZ of one of
the plasmids are identified as white colonies on X-gal.  Recently, I
tried Quiagen prepped DNA in this assay.  I generated a lot of white
colonies and thought I had a "transposition Bonanza".  It turns out
that the plasmids isolated from these colonies were the products of
recombination, unrelated to transposition.  This is only a problem with
Q. prepped DNA.  My hypothesis is that the Q. preps generated double
stranded breaks, which then stimulated homologous recombination between
plasmids.  This may account for your lack of transfection if
recombination can alter your selectable marker, for example.  That is,
you may be getting transfection, but you just can't detect it.

Of course, take this for what it's worth.

Ive gone back to Cs preps, for now anyway.

Let me know if this makes any sense in your case.

Ken saville 




More information about the Methods mailing list