fuzzy bands on long seq gel?

deborah_hailstones_at_emai at smtpgwy.agric.nsw.gov.au deborah_hailstones_at_emai at smtpgwy.agric.nsw.gov.au
Thu Feb 12 18:24:37 EST 1998


     David
     In my experience, fuzzy bands always relates to running the gel at too 
     high a temperature... i would presume that as your resistance drops in 
     a longer run the gel is heating up...
     air cooled sounds good, otherwise drop the voltage further and be 
     patient!  havent ever tried formamide in the gel.. i used to use wedge 
     shaped spacers to stack the bases up at the bottom of the gel.
     Deb


______________________________ Reply Separator _________________________________
Subject: fuzzy bands on long seq gel? 
Author:  <dmachugh at dux4.tcd.ie (David MacHugh) > at smtpgwy
Date:    2/12/98 5:39 PM


     
Hi,
     
We have been banging our heads against the wall trying to eliminate fuzzy 
bands from manual sequencing gels, particularly with long runs or very 
long runs.
     
We are direct-sequencing a 750 bp PCR product using biotin-labelled 
primers and the streptavidin-coated magnetic beads to make single-stranded 
template.
     
The sequencing gels are loaded on standard BRL S2 rigs with 6% Sequagel 
acrylamide and 1XTBE including ammonium acetate (~0.6 M) in the bottom 
buffer tank to compress the bands near the bottom of the gel.
     
Short runs of the gel (approx 3 hrs) present no problems and the bands are 
clear and easy to read.  When longer runs are attempted (in order to get 
the whole PCR product using primers from either end), usually of about 5-6 
hrs, the bands start to become fuzzy and difficult to read.  We have tried 
running at a lower voltage to no avail and are going to try the new 
air-cooled BRL S2001 system.
     
Does anyone have any other suggestions how these long run gels could be 
improved? 
     
I have heard that increasing the buffering capacity of the TBE (perhaps 
1.5-2.0X TBE) may improve the clarity of the bands, but we have yet to try 
this.  Would the inclusion of formamide in the gel improve the
resolution?  Should we increase the acrylamide concentration? etc...
     
Any comments or suggestions gratefully accepted!
     
Thanks in advance.
     
     
David.
     
David MacHugh,
Genetics Department,
Trinity College,
Dublin 2.
     
     





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