Frowns in polyacrylamide gel - (Dec/10/2006 )

Hi,

Was running thru the archives for solutions to my current vertical denaturing polyacrylamide gel problem. However, I'm putting in PCR products into it ("conformation sensitive gel electrophoresis"). Hope tis ok I gate-crash into this forum.

Instead of smiles, I've frowns or inverted smiles. My running conds are 800V for ard 4hrs at room temp. During the initial electrophoresis period, the glass plates are warm to touch esp at the centre (abt 37C). The bands become slightly 'frowny'. As the run progresses, the plates become cooler and the bands are really seen as 'frowns'. And I do notice that the bottom wire (of the old sequencing electrophoresis chamber) is shorter than the top one.

Any comments guys?
Thanks!

p/s
had actually posted this in another topic under the Protein and Proteomics forum last week. Not getting any feedback, hence this fresh attempt.

-billybong-

Why don't you let it run at 4°C? 800V is relatively high. Try it! Or try to cool it otherwise.

-biomaus-

QUOTE (biomaus @ Dec 11 2006, 08:33 PM)

Why don't you let it run at 4°C? 800V is relatively high. Try it! Or try to cool it otherwise.

So, you think it's the heat that's causing the frowns?

A few published papers of CSGE used 40W for > 4 hours (depending on PCR product size) at room temp. Which means, the gel would be much warmer, don't you think so, biomaus? But strangely, their bands were ok.

Thanks, biomaus for your feedback

Btw, anybody reading this who's used or who knows someone using the CSGE method for mutation detection?

-billybong-

I agree, 800V is pretty high...how big are the gels you are using? We use small ones, and only run at ~150V max.

-Cheamps-

QUOTE (Cheamps @ Dec 13 2006, 04:50 AM)

I agree, 800V is pretty high...how big are the gels you are using? We use small ones, and only run at ~150V max.

hi there,

my gel is of 330x390 mm. are you running sds-page gels, cheamps?

-billybong-

QUOTE (billybong @ Dec 14 2006, 04:04 AM)

QUOTE (Cheamps @ Dec 13 2006, 04:50 AM)

I agree, 800V is pretty high...how big are the gels you are using? We use small ones, and only run at ~150V max.

hi there,

my gel is of 330x390 mm. are you running sds-page gels, cheamps?

This is a large gel, I used to do bigger ones for sequencing. I can give you a few tips.

First, heat is caused by the current (amperage), not the voltage, and that depends on the buffer you're using. You can have 800V with low current (if the buffer is low salt) hence less heat. Try a TBE buffer at 0.5X (1X will give more heat at 800V), it's suited for such big gels.

Also, keep an eye on the amperage. And, make sure the gel is well polymerized before using. I used to leave it polymerize for 1-2 hours.

So, overall, to avoid frowning, use appropriate buffer, keep amperage low, and let polymerize.

Good luck.

-9mm-

much thanks for your tips!
but how low shd the amperage be to prevent heating up? am actually using an old sequencing chamber for CSGE.
am using TTE buffer btw. any comment on it with regards to its usage for a big gel?

-billybong-

QUOTE (billybong @ Dec 20 2006, 11:07 PM)

I have never worked with TTE buffer. Heating up is unavoidable actually, as long as it's within acceptable limits. I remember reaching about 50C on our gels without any frowns. Is it possible the TTE buffer isn't as suitable as TBE buffer?

I suggest you try TBE buffer. Also, I hope you're using the TBE/TTE buffer when you prepare the polyacrylamide gel !!!! If you're using a buffer different from the migration buffer, you will have some serious frowning effects. Double check that.

-9mm-

-billybong-