DNA sequencing gels

William R. Morgan wmorgan at ACS.WOOSTER.EDU
Tue Jul 20 11:33:10 EST 1993


Bob Karn:

After you pour, are you clamping the top of the sequencing plates using a
top clamp(s) and spring clips.  In a supplemental instruction sheet (BRL's
instruction form #14219), BRL recommends placing at the top "two spring
clips over the (side) edge of the plates to clamp on the comb...( Do not
place the pressure point of the spring clip over the side spacer.)  Place
the top clamp over the top of the glass plates and clamp the center of the
top edges of the plates securely against the comb.  They further note that
"if spring clips are not put in place during polymerization, the comb(s)
will not fit tightly, which increases the possiblity of leaks between
wells"!  

This clamping procedure isn't emphasized very well in the Instruction
Manual that accompanies the S2 apparatus, but is described (and
diagrammed!) nicely in the form #14219 instructions (entitled "Instructions
for preparing sequencing gels with BRL's sharkstooth combs, gel sealing
tape, and top clamp").  If you don't have this form, I suggest you request
that BRL FAX it to you pronto.  If they can't, I can FAX you my copy.

By the way, are you still using gel sealing tape to seal your gel plates? 
I haven't used tape in years.  Let me know if you're interested in how you
can avoid this tedious task.

I hope this solves your problem,
Bill Morgan


>Dear Sequencing Netters:
>        I have relatively little experience running DNA sequencing gels and
>am having problems with samples spreading across other lanes when I am loading
>them.  I use a BRL S2 gel of the standard 0.4 mm thickness and the usual
>concentrations of gel components and urea.  It seems that I have this problem
>more often (but by no means soley) when I pour the gel the night before and
>run it the next day (I have been led to believe that this practice is accep-
>table if one takes measures to keep the gel from drying out while sitting
>overnight).  The spreading problem does not seem to be due to the shark's
>tooth comb penetrating the gel to any particular degree.  Sometimes I get
>very good loading (i.e. no spreading across lanes) and sometimes gross cross-
>lane contamination - and I can see no pattern to why it works well sometimes
>and not others.  I would be most grateful for tips from you oldtimers in the
>sequencing game.
>Bob Karn
>KARN at BUTLERU.edu

William R. Morgan
The College of Wooster
Department of Biology
931 College St.
Wooster, OH  44691
Phone:  216-263-2026
FAX:    216-263-2378
E-mail: wmorgan at acs.wooster.edu




More information about the Methods mailing list