Prepping Plasmids for Automated Sequencing?

Jens Alfken alfken at zedat.fu-berlin.de
Sat Jan 4 12:00:32 EST 1997


In article <martinez-0301971533420001 at mac20.agoff.umn.edu>
martinez at puccini.CRL.umn.edu (J. Pat Martinez) writes:

> Any suggestions on miniprep columns for purifying plasmid DNA to be used
> for automated sequencing?  I was considering buying the mini prep spin
> columns from Qiagen or the perfect prep columns from 5 prime 3 prime.
> 
> Pat M.


I have been using both the Quiagen spin columns and their vacuum setup
in order to generate plasmid DNA for sequencing with a LICOR machine.
One problem I and others in the lab have, is residual ethanol that will
inhibit the sequencing reaction. The main reason seems to be that after
years of usage the «sponge« of the vacuum manifold has become stiff and
will no longer give an adequate seal. The resulting low vacuum does not
seem to be good enough to allow sufficient drying of the samples before
elution. 
I had good results with both the Quiagen spin columns and the Promega
vacuum system (Wizard). 
On the other hand I have colleagues that get decent results with an old
fashioned alkaline lysis protocol using no kit etc. 

Jens Alfken

alfken at zedat.fu-berlin.de



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