Protocol for Re-using of Qiagen tips

Ferland Louis H. ferlandl at ERE.UMontreal.CA
Wed Mar 20 03:26:30 EST 1996


Harry, 

I would go for your guess #1. I had the same problem of too much muck in 
the culture to generate the large quantities of some of my plasmids. I 
solved the problem with the good old chloramphenicol amplification trick: 
I inoculate 500 ml of LB in the morning, add the chloramphenicol (I 
can't recall the concentration from the top of my head, but it was pretty 
standard) when the OD at 660 nm is around 1.0-1.2 (usually 3.5 to 4 hours), 
and let grow further overnight. That gives anywhere between 1 and 3 mg of 
plasmid that can be purified on a Qiagen Mega column without any problem 
of slow elution.

Hope this helps,

Louis


On Wed, 13 Mar 1996, Harry J. Witchel wrote:

> Date: Wed, 13 Mar 1996 13:35:07 GMT
> From: Harry J. Witchel <Harry.Witchel at bristol.ac.uk>
> To: methods at net.bio.net
> Subject: Re: Protocol for Re-using of Qiagen tips
> 
> Mads --
> 	I routinely have a problem with Qiagen maxi preps in that the columns 
> flow very slowly, as if they were clogging.  To run 30 mls through a column 
> might take 3-4 hours on a bad day.  To reduce this problem, I have taken to 
> respinning the initial KOAc supernatant a second time a high speed, and then 
> to run the entire supernatant through a .45 um syringe filter before loading 
> onto the column.  Even so, the column sometimes (unpredictably) slows down.  
> 	My two guesses would be that:
> 	
> 	1)  Too much bacterial material in starting material (I solubilize a 
> 250 ml overnight culture of Terrific broth into 10 ml P1, then add 10 ml P2 
> and P3).
> 
> 	2)  I let the column run dry after wetting it with QB.  Usually I load 
> just when QB is running out, but sometimes there might be 10 minutes where the 
> top of the column is dry.  Qiagen claims that their column design should 
> prevent the actual matrix itself from drying out.
> 
> Do you have any guesses?
> 	Thanks, 
> 	Harry
> 
> Harry.Witchel at Bristol.Ac.Uk
> 
> 

Dr. Louis H. Ferland
Centre de Recherche, Hotel-Dieu de Montreal
Dept de Nutrition, Universite de Montreal
Phone: (514) 843-2757     FAX: (514) 843-2719




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