Wedge gels (Salt gradient gels)

Dennis J. Templeton djt2 at po.CWRU.Edu
Thu Aug 12 12:20:33 EST 1993


In a previous article, Klaus.Matthaei at anu.edu.au () says:

>>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:
(deleted stuff
>>> 
>>> 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,
>>> dennis
>>>   
>>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?
>
>
>If you run constant POWER then you should not overheat at the top of the
>gel.  This will slow things down a little though.  I add a 1/3rd volume of
>1M NaAc at the time of loading and causes compression of the BPB to XyC
>area to about half.  My sequencing gels therefore are run to XyC 60cm, BPB
>80cm instead of the normal 40 - 60cm respectively.
>
>Cheers, 
>
>KLaus

Klaus is somewhat off the mark here; if your run your gel on constant power
the *total* heat produced will be constant, but because the resistance is
much lower at the bottom of the gel, almost all of the heat is now produced
at the top, leading to *local* overheating.  This is true for wedges and
other electrolyte gradient systems too.  We learned this the hard way.

Our normal protocol for 6% gels, 45 cm in length is to pre-run and run at 2
kV for about 90 minutes, maybe 2 hours, then add the salt and lower the
voltage to 1700V, with a fan directed at the top of the plate.  Our gels
have a buffer backing, but a metal plate backing is probably equivalent.
The top of the gel gets slightly warmer this way, and the bottom cools off.
 We continue electrophoresis for an extra 2 hours, or until the tech wants
to go home, then fix and dry on the plate as has been discussed here ad
nauseum. 

I wouldn't try lowering the salt and running longer, the net effect would
be that the heating comes up slower, and your BPB might runn off with the
small bands. Instead, I would lower the voltage until there is no problem
with heating.

Which reminds me of a story (true) about when Dan Donoghue at UCSD ran 2
meter plates made from pyrex at 10 kV to resolve a compression.  The gel
got so hot it boiled in situ, resulting in a dramatic pyrotechnic display
(fortunately sealed behind the plates) that melted pits *in the pyrex*.
Don't let this happen to you.

have fun
dennis



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