DNA prep for transient transfection

Curt Ashendel ashendel at aclcb.purdue.edu
Fri Feb 17 12:39:54 EST 1995


On 17 Feb 1995 16:13:39 GMT, 
Martin D. Leach  <leach at bu.edu> wrote:
>
>Always check the amount of nicked bands prior to transfection...if there
>appears to be a lot (10-15%) then the batch of plasmid is not used for
>transfections.

Actually, contrary to the implication of the above statement, the state of 
supercoiling of plasmid DNA has no effect on transfection efficiency.  
This is demonstrated by the fact that many people get equally high levels 
of expression after transfection with either supercoiled or with linearized 
plasmid DNA.

The key to high efficiency is the purity of the DNA.  The major 
contaminants that interfere are RNA and DNA (surprise!).  ss(oligo)DNA  
heavily contaminates most plasmid preps (but the extent of such 
contamination depends on the prep method, the particular plasmid size, and 
frequently on non-controlled prep parameters).  This contamination 
cannot be easily seen on gels, but has a large effect on expression levels 
after transfection. I have found that CsCl banding of DNA a single time 
only removes some of this type of contaminant, and that for some plasmid 
preps, much higher transformation efficiency can be obtained it the plasmid 
DNA is banded on EtBr-CsCl a second time. 

Just my $0.02 worth. YMMV.

Curt Ashendel
Purdue University
West Lafayette, IN
ashendel at aclcb.purdue.edu



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