Wedge gels (Salt gradient gels)

Klaus.Matthaei at anu.edu.au Klaus.Matthaei at anu.edu.au
Fri Aug 13 04:02:36 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

Hi Denis

Nice theory but in my experience the top of the gel hasn't overheated.  I
have the Biorad temperature sensitive indicators (one top, one bottom) and
after the addition of the acetate the temperature never goes above the
required 50*C.  I do however use Biorad IPC setup which has a very good
cooling buffer tank on the back of the gel.  Maybe this gets rid of the
problem that you propose.  I will be interested to hear more.

Cheers,

Klaus 
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Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"If at first you don't succeed - Get a bigger hammer"
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