DNA Quality and Transfections - does it really make a difference?

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Mon Mar 1 16:32:03 EST 1999


Hi All:

This has been an interesting thread as it comes up every now and then.   I
think it will boil down to what works for you, the individual investigator,
is what is best.  

While in a previous locale, I was testing plasmid purification methods as a
means to assess what gave the best for transfection.  What I was using was
a beta-gal that was prepared 4 different ways.  All other variables were
kept constant as best they could be.  The DNA was electroporated into COS-1
cells and all transfectants were grown in the same media, etc., etc...

The plasmid was prepared via: 1) Qiagen mini columns; 2) Promega wizzard;
3) Alkaline lysis-PEG mini prep (BioTech. 14:532), and 4) standard CsCl
single-banded prep.    All DNA was preped within a day of each other and
transfections were done on the same day.  48 hours later, the cells were
processed for beta-gal activity as described in the Harvard manual.

"For me" (note the quotes!), there was no question that CsCl was by far the
best with about 60-70% of the cells turning blue.   In second place was the
Alk-Lysis PEG mini; third, the Wizzard, and last was the Qiagen mini-prep.
 To try and rank the 4 protocols on a scale of 10 to 1 with 10 being the
best, CsCl-10, Alk-Lysis PEG-7, Wizzard-3, and Qiagen-1.    There was quite
a bit of difference between the PEG mini and the Wizzard.

So if asked by my colleages in the hall, my response will be CsCl.   But
that's me, based on my experience.  Others out there were to try these four
methods, may in fact come up with a markedly different order of performance.

To each his own...
David
=============================
 David L. Haviland, Ph.D.
 Asst. Prof. Immunology 
 University of Texas - Houston, H.S.C.
 Institute of Molecular Medicine  
 2121 W. Holcombe Blvd.  
 Houston, TX  77030 
 Internet:"dhavilan at imm2.imm.uth.tmc.edu" 
 Voice: 713.500.2413  FAX: 713.500.2424
 ------------------------------------------------------  
I try to take one day at a time but lately several days
have attacked me at once!
=============================



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