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denature gel shrink, help


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#1 Tintin

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Posted 05 August 2009 - 07:56 AM

I run a denature gel for my northern blot. The gel was made in gel running buffer (50mM boric acid, 1mM sodium citrate, 5mM NaOH, pH7.5) and 0.3M formaldehyde.
After I loading the RNA and turn on the electrophoresis (~9V/cm), I found that the dye running faster than I did before. 2 hours later when I recheck my gel, all dye has running out (it normally should take 3-4 hours to run to the end). More strangely, the gel shrink and shape changed. What's wrong?

PS:
My gel running buffer was made 12 months ago and kept on bench in room temperature. I haven't used it for one year, but it's clear.
I made a 18.5% formaldehyde stock solution from paraformaldehyde freshly. But I do add some 1M NaOH (45ul to 5ml paraformaldehyde) to help it dissolve, according to a CHIP protocol. After I made this solution, I found PH 11, then I add HCl to neutralize it to 7.5.

#2 mdfenko

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Posted 05 August 2009 - 08:26 AM

did you check the pH of your running buffer? it may have changed during the year at room temperature.

also, your buffer may have increased in concentration (by evaporation).
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#3 Tintin

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Posted 05 August 2009 - 10:04 AM

yes, I adjusted it. So PH can make the gel shrink? I met this once before when I made a TAE gel, which I mistook water instead of TAE to make the gel.

#4 mdfenko

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Posted 05 August 2009 - 11:02 AM

the pH and the buffer (salt) concentration will affect the speed of migration, the concentration, if higher, can cause the shrinkage.
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#5 Tintin

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Posted 05 August 2009 - 01:51 PM

the concentration of the running buffer or the buffer in gel? I used the same thing. High PH won't shrink it? I'm worried about the PFA. I used NaOH in it.

#6 mdfenko

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Posted 06 August 2009 - 10:07 AM

i don't think you used enough naoh with your pfa to make a difference in the final solution.

i think that your buffer may be off pH and at a higher concentration than you expect.

this leads to the difference in mobility and may also cause the gel to overheat.

the higher concentration (can cause shrinkage of the gel) and heating may be the reason for the shape change.

what is the shape of the gel?

any pictures?
talent does what it can
genius does what it must
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