DNA prep for transient transfection

QIAGEN QIAGEN at kaiwan.com
Fri Feb 24 14:05:27 EST 1995


In article <58176.ashendel at aclcb.purdue.edu>, "Curt Ashendel"
<ashendel at aclcb.purdue.edu> wrote:

> 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. 
> 


Dear Martin and Curt,

It is true that plasmid purity is extremely important for transfection
efficiency, but there's a much easier way to purify it than CsCl. Ehlert et
al. (Biotechniques 14: 546) showed that DNA purified with QIAGEN
anion-exchange resin gave transfection efficiencies comparable to those
obtained with DNA purified 2X with CsCl. QIAGEN resin-based products are
available in a variety of different formats to accomodate a range of sample
size and throughput requirements. Not only is the DNA purified with QIAGEN
resin as pure as 2X CsCl, you can do as many as 96 minipreps, or a smaller
number of larger scale preps, in 2 hours or less. Call for a free catalog.

Brian Taylor
Product Manager 

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QIAGEN       800-362-7737
Temporary Email Address          qiagen at kaiwan.com



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