harvesting DNA from a lambda gt11 clone

[QIAGEN]

In article <1995Jul13.152951.2601 at vms.huji.ac.il>, benziman at vms.huji.ac.il
(patricia) wrote:

> I am trying to obtain a clean lambda gt11 DNA prep. for sequencing without any success. 
> I used Quiagen kit but the yield was low and the ratio 260/280 was always
> less than 1.7. I also tried the Maniatis method using SDS lysis, with no
> great success. Does anyone have a suggestion? Thanks. Patricia.

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