DNA purification of lambda gt11 clones

[QIAGEN]

In article <3uagun$fdn at shum.cc.huji.ac.il>, patricia ohana
<benziman at vms.huji.ac.il> wrote:

> I am trying to purify DNA from a single lambda gt11 clone in preparation for
> direct sequencing. Using the Qiagen kit my yields have been low and the
> OD 260/280 ratio is less than 1.7. I have been using plate lysates as starting
> material, using about 10 plates containing confluent lysis.
> Does anyone have any suggestions?

Dear Patricia,

One possible explanation for your low yield and low 260/280 ratio with your

QIAGEN lambda prep is overloading. If an excess of lysate is loaded onto
the 
column, contaminants such as capsid proteins compete with the DNA for
binding 
sites on the column and yield is reduced. This can also result in low
260/280 
ratios, since the ratio of DNA to protein and other contaminants may be 
affected. I recommend that you try titrating the amount of material loaded
on 
the column to find an optimum amount for your kit size. For a mini column, 
10 ml of lysate is the maximum. Other possibilites for the source of your 
problem include SDS carryover, which can block DNA binding to the QIAGEN 
resin, and use of agar instead of electrophoresis-grade agarose in your 
plates. 

Brian Taylor
QIAGEN, Inc.  
-- 
QIAGEN       800-362-7737
Temporary Email Address          qiagen at kaiwan.com