IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

peak stretching

Maureen Maureen
Fri Feb 7 12:44:40 EST 1997


> Re: Peak stretching around 200bp
> 
> demma at welchlink.welch.jhu.edu
> Fri, 7 Feb 1997 06:50:15 -0800
> 
>    * Messages sorted by: [ date ][ thread ][ subject ][ author ]
>    * Previous message: tomlinson at pplros.demon.co.uk: "Re: Anyone
>      having problems with PCR Blunt?"
> 
> ----------------------------------------------------------------------
> 
> Date: Fri, 7 Feb 1997 06:50:15 -0800
> 
> Message-Id: <199702071450.GAA25748 at net.bio.net>
> 
> To: autoseq at net.bio.net
> 
> Subject:     Re: Peak stretching around 200bp
> 
> From: charlie.demma, demma at welchlink.welch.jhu.edu
> 
> In article <5cnrkv$icn at net.bio.net>, Dr Tim Sawbridge,
> T.Sawbridge at forbio.com.au wrote:
> 
> > Dear Sequencers,
> > we have been seeing stretching of peaks from around 190
> > to 210 bp across the gel on a 377 with resulting loss of resolution.
> The
> > peaks return to their normal width after this so it seems to be a
> > localised problem at around these fragment sizes. We are doing 3.5
> hour
> > runs on 36cm plates using dye-terminator chemistry with the ABI kit
> on
> > double stranded plasmid DNA. Has anyone seen this and know what
> might be
> > causing it?
> > Thanks in advance
> > Tim Sawbridge
> >
> > T.Sawbridge at forbio.com.au
> 
> We have been having the exact same problem (377, DT, 36cm, 4X run). We
> thought it was a problem in the Gel reagents but it continued after
> changing them. We acid cleaned the plates but that didn't help.It
> happens
> on both a 4% polyacrilimide gel and on the Long Ranger gels. We
> usually
> pour our gels in the evening and run them the next morning. Maybe
> these
> clues will help. Please respond with some help if you figure this out.
> 
> Thanks, Charlie Demma Demma at welchlink.welch.jhu.edu
> ----------------------------------------------------------------------
> 
>    * Previous message: tomlinson at pplros.demon.co.uk: "Re: Anyone
>      having problems with PCR Blunt?"


We have traced the aberrations in this region of the gel to old or 
improperly 
de-ionized formamide used in preparation of the loading dye. We now store 
the 
formamide (Amresco) over Biorad's AG501 resin (1g/100ml) at 4 C, replace 
this stock 
bottle every 3 months and make fresh formamide/dye mix for each day's 
runs. It may be 
overkill but we haven't seen any loss of signal or resolution in the 
150-250 nt 
region of the gel since adopting this practice.


-- 
Maureen Dolan, Ph. D.
Department of Molecular Biology
Massachusetts General Hospital
Wellman Bldg.  1131
50 Blossom Street
Boston, MA 02114

Office: (617) 726-5930 	Lab: (617) 726-5938	FAX: (617) 726-6893



More information about the Autoseq mailing list

Send comments to us at biosci-help [At] net.bio.net