Transfections using QIAGEN

Amos Heckendorf nestgrp at world.std.com
Mon Mar 27 06:34:01 EST 1995


In article <QIAGEN-240395104654 at 192.0.2.1>, QIAGEN at kaiwan.com (QIAGEN)
> Kim Budelier
> QIAGEN Inc.  wrote:
> Culture volume: When alkaline lysis conditions are optimal, P2 lyses all
> the cells and P3 precipitates quite a bit of protein and all the gDNA. The
> column procedure then removes the remaining protein, etc. 
> If too much culture is used, the efficiency of alkaline lysis is reduced
> and the column is overloaded. All the protein and polysaccharides may not
> be removed in the wash steps and find their way into your final eluate. 
> You should be able to avoid these contaminations by using the recommended
> culture volumes or growing to the proper cell density (generally, 12-16 hrs
> growth should be o.k.). Also, we do not recommend rich broth (TB, 2xYT) as
> this increases your cell density 4-5 fold and the recommended volumes of
> alkaline lysis buffers would not be optimal.
> 
> Capacity of column: the nomenclature of the column tells you the binding
> capacity (e.g. QIAGEN-tip 500 binds 500 µg). I have talked to many users
> who thought the number was indicating the culture volume to process.
> Actually, using only 100 ml LB culture will yield approx. 500 µg of a high
> copy number plasmid. 

I don't agree with the explanation of column overload.  Since the plasmid
(barring any genomic contamination) is the last ionic species to elute
then it will displace all other lesser bound species as it loads up the
column.  The NucleobondAX chemistry which your sales people have been
telling customers is the same as the Qiagen chemistry and which is also
rated according to the nomenclature of the column (e.g. Nucleobond AX 500
binds 500 µg) seems to be able to bind DNA to capacity regardless of
culture volume or DNA concentration. Is this a result of different
chemistry or different concentration of applied solution? 

I think what you and Martin are dealing with is a case of viscosity and a
lack of difusional control.  When you have the excess carbohydrates or
proteins in there you quite possibly are having viscosity problems
inhibiting diffusion to your surface chemistry.  While this isn't
necessarily a negative thing it does require your customers to compensate
for the problem by having to underload the column, or conversely to dilute
the sample in order to get the optimal performance from your product.

Keep up the good work, I appreciate your comments and insights.

-- 
Amos Heckendorf 
(nestgrp at world.std.com) 
Tel: 800-347-6378 or 508-481-6223; FAX: 508-485-5736.  



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