IUBio

Terminator residues

Jack Leonard Jack.Leonard at amicon.com
Fri Oct 25 09:23:50 EST 1996


> Terminator residues.
> 
> Cameron McCrae (Cameron.McCrae at anu.edu.au)
> Thu, 24 Oct 1996 09:53:09 +1000 (EST)
> 
>    * Messages sorted by: [ date ][ thread ][ subject ][ author ]
>    * Previous message: Andrea Schmiechen: "Funding For Sequencing"
> 
> ----------------------------------------------------------------------
> 
> Message-Id: <199610240857.BAA29400 at net.bio.net>
> 
> To: autoseq at net.bio.net
> 
> Date: Thu, 24 Oct 1996 09:53:09 +1000 (EST)
> 
> From: Cameron McCrae <Cameron.McCrae at anu.edu.au>
> 
> Subject: Terminator residues.
> 
> Morning All,
> Does anyone know of a reliable method for eliminating the
> characteristic dye 'blobs' from the beginning of a dye terminator
> extension. It seems that simply following the ABI protocol results in
> quite
> variable amounts of excess terminators even though we, and all of our
> users, have been using the FS kits since their release.
> 
> Thanks in advance,
> 
> Cameron
> 
> ******************************************************
> Cameron McCrae
> Biomolecular Resource Facility
> Australian National University
> GPO Box 334
> Canberra ACT AUSTRALIA
> Phone No. <61> <6> 249 4326
> Fax No. <61> <6> 249 0735
> E-mail Address Cameron.McCrae at anu.edu.au
> ******************************************************
> ----------------------------------------------------------------------
> 
>    * Previous message: Andrea Schmiechen: "Funding For Sequencing"


Good morning,

Spin columns seem to remove dyes "blobs" reproducibly.  There are the
popular and commercially available "CentriSep" spin columns, but the
modified characteristics of Taq-FS has allowed many to switch to EtOH
precipitation to save money.  

The drawback with EtOH precipitation is that you see re-emergence of dye
"blobs", and sometimes even T "blobs" later in the sequence.  Recently,
ABI (Dr. Chen; chensm at ccmail.apldbio.com) developed an improved EtOH
precipitation for Dye-Terminator removal.  Treating the reaction with
shrimp alk. phoshatase before EtOH precipitation evidently eliminates
dye "blobs".  We have found that calf intestinal alk. phosphatase also
works.  I believe that dephosphorylation works because once you remove
the negatively charged phosphates from the unincorporated
Dye-terminators they will no longer migrate into the gel (remaining base
and dye carry positive charges if anything).

Dr. Chen will probably be happy to e-mail you his protocol.

-Jack-
-- 
Jack T. Leonard, Ph.D.
Manager, Lab Products and Applications
Amicon, R&D





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