SDS-PAGE Pouring Problem

[Katatonic]

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I figured this would be the best place to post this question in case any of you have experienced this. I work in a lab making SDS-PAGE gels for the researchers but I've been having a lot of problems lately, and neither me nor the head researcher can figure it out. I use a custom kind of box they made to pour 13 gels at a time, filling a trough from the bottom from a gradient mixer (these are 12-17% I'm having problems with). The box has an open top and the plates are stacked (small plate)(spacers)(big plate) for 26 plates total with a wedge in the back to keep them tight.

In roughly half of the gels after pouring the first phase, before the stacker, they dip off to the side from a little bit to very sharply. This has led to me having to throw away 50% of everything I make which is a large waste of materials. Has anyone experienced this "dipping" off to the sides of their interfaces? If so, how did you fix it? Thanks!

 

[Doctor&Geek]

If you're using those BioRad multistackers, most of the time, this occurs due to leakage. Check to make sure you don't add too many OR too little plates to the multistacker.

 

[Katatonic]

I'm not sure what you mean by a "multi-stacker". I mix up the stacker solution myself and pour it over the top of the plates right before inserting the combs. The leaking that we've suspected is due to some of the spacers not being flush with the side of the box.

 

[Calf]

I've never used a multistacker either. I've only poured single gels. If you can't fix it then 50% waste might make it worth cutting back on the number of gels or pouring them one at a time...

 

[gbwillner]

This does sound like a leakage problem. do you place your plates in a plastic bag to contain possible leaks?

 

[Doctor&Geek]

I'm not sure what you mean by a "multi-stacker". I mix up the stacker solution myself and pour it over the top of the plates right before inserting the combs. The leaking that we've suspected is due to some of the spacers not being flush with the side of the box.

I'm talking about one of these:
http://www.bio-rad.com/LifeScience/pdf/Bulletin_4006193A.pdf

 

[seraph524]

Invitrogen Novex Pre-Cast gels ftw!

 

[Doctor&Geek]

Invitrogen Novex Pre-Cast gels ftw!

GIMME THE CASH!!!

[YOUTUBE]http://www.youtube.com/watch?v=OADJl-CVDo0[/YOUTUBE]

 

[jlamacc1]

Invitrogen Novex Pre-Cast gels ftw!

They're so smooth and silky!

 

[FSAP]

Ew, you make your own gels?

 

[Katatonic]

Ew, you make your own gels?

Haha yes, but I'm not complaining since it is my the sole purpose of my job. Thank you all for the advice. I'm almost sure it's either a leaking problem, OR something to do with the n-butanol I'm adding on top of the gradient while it polymerizes for 2 hours. It dawned on me yesterday, that while I was sucking up the almost-depleted amount of n-butanol with my pipet (it's water saturated), I could inadvertently be getting regular water in with it since the phases were so close to each other. If this were happening, it would allow oxygen to diffuse into the gel, completely screwing up polymerization of the acrylamide. Hopefully I figure it out soon, as things have gotten worse this week and I'm getting an average yield of 42.3%!

 

[tco]

If you're adding butanol to the top, make sure you add it very slowly. We had a problem with it remaining in the gel while it polymerized(not quite!)...For whatever reason?

 

[Katatonic]

If you're adding butanol to the top, make sure you add it very slowly. We had a problem with it remaining in the gel while it polymerized(not quite!)...For whatever reason?

What do you mean slowly? I add 200 microliters in the center of the plate to let it slowly spread across the top of the gel. Should I wait a minute before adding it? I figured that would leave it exposed to the oxygen for too long, but I'm not sure. The gels have gotten slightly better the last 2 days I've worked. Hopefully they keep improving to where I can keep most of what I make haha.