Tim Dunn t.dunn at mailbox.uq.oz.au
Wed Jun 21 19:04:42 EST 1995

In article <jr.6.0009E78F at dna.bio.warwick.ac.uk>, jr at dna.bio.warwick.ac.uk

> Dear all,
>                I recently did a plasmid prep using the QIAGEN megaprep
kit and 
> obtained DNA with a O.D.(260/280) ratio of 1.7. Having decided to try and 
> clean it up further, I ppt'd the DNA using 2 vols of ethanol (including
> followed by 2 washes with 70% EtOH. This time my O.D.(260/280) ratio actually 
> went down to 1.65. Upon re-ppt'ing the ratio went down to 1.6. (The actual 
> amount of DNA recovered is the same.)
>         So does anyone know why the O.D (260/280) ratio is decreasing
everytime I 
> re-ppt the Qiagen prep'd DNA and how do I rectify the problem? 
>         Thanks in advance.
> Jaz

EtOH ppt'ing also brings down any protein contaminating the prep.  You
loose a small amount of DNA each time you ppt, depending on the DNA
concentration.  Reprecipitating plasmid preps is good for getting rid of
organic contaminants like phenol, salt etc, but it won't separate your DNA
from protein, maybe you need a nasty step like phenol extraction.  We have
found the new type of Qiagen columns (500s) are able to cope with much
less culture volume than the older type, ie. there is MUCH less resin in
the column. If we use more culture than is recommended in the protocol,
the plasmid prep quality plummets, (I'm not sure if this is relevent!) 

I have a question about Qiagen preps also.  A while back I was denaturing
plasmids and binding to nylon filters for nuclear run-on assays.  When I
used NaOH to denature Qiagen plasmid preps, I got lots of gloop coming out
(clear jelly-like stuff).  I spun this stuff out and lost only a small
amount of plasmid, BUT has anyone seen this stuff also?  It is not present
in CsCl preps.  What is it?? 


Tim Dunn
Centre for Molecular and Cellular Biology
University of Queensland
Australia 4075
e-mail t.dunn at mailbox.uq.oz.au
phone  (07) 365 4565
fax    (07) 365 4388 

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