In article <24b9l4$s87 at usenet.INS.CWRU.Edu>, djt2 at po.CWRU.Edu (Dennis J. Templeton) writes:
>> In a previous article, jwelch at acsu.buffalo.edu (John J Welch) says:
>> >Dear Net:
> > I am interested in sequencing/footprinting on relatively small
> >DNA sequences. A 20% denaturing PAGE gel will accomodate almost the entire
> >sequence, but the smallest fragments usually get very spread-out and the
> >very smallest zip right off the end of the gel. One person suggested
> >pouring a gel of variable thickness to create an electrical field gradient.
> >Large pieces would migrate faster, small pieces would migrate slower, relative
> >to their mobilities in a standard "flat" gel. This makes sense to me, and
> >I have seen examples of such gels. After checking searching the archives
> >for this group, I have seen nothing on this topic except a reference to
> >thermal gradient gels which may yield similar results.
> > I would like to see if anyone has had experience with E-field
> >gradient gels, or if anyone can give pointers to literature on the subject.
> >According to the person who recommended this technique, his lab used to
> >home-make the spacers out of nylon plastic sheets using a lathe. Can
> >anyone recommend a commercial supplier?
> > Thanks in advance for information. Please respond by Email to
> > welch at sc3101.med.buffalo.edu> >
> > Or reply via this newsgroup.
> >John Welch
> >Roswell Park Cancer Institute
> John, we have used both wedge gels and salt gradient gels for sequencing.
> While I am sentimental about the wedge, having developed and popularized it
> years before it was eventually published, it doesn't work as well as the
> NaAc salt gradient described below (I haven't the reference at hand)
>> Pour and pre-run a regular urea gel in 0.5x TBE (55mM Borate= 0.5x) using
> the same buffer for gel and electrolyte buffer.
>> Pre-run, load, and run the gel as normal, until the BPB (fast dye) reaches
> to within a couple of inches of the bottom.
>> Add to the bottom chamber 1/2 volume of 3M NaAc pH 7 (i.e. final conc about
> 1 M Na.)
>> CAUTION: the top part of the gel will now heat more than the bottom.
> either cool the top with a fan, or lower the voltage, or both.
>> Continue electrophoresis as long as desired. The BPB will essentially
> *never* run off the bottom, and the XCFF will get to within 4-6 inches of
> the bottom. That's when we stop it for sequencing. This yields an extra
> 80-100 NT from each gel, and it is well worth the slight trouble.
>> good luck,
>I am interested in this protocol, but there is no way I can cool my system, so
I need to know how hot the plates get to make sure they survive!
Is possible to compromise using less NaAc if required due to temp. and still gaining
resolution, and how much longer do you run your gels with the salt gradient compared
to standard gel runs?