MS research- stupid gel problem

[NYyanx28]

So, for the past few days I've been having an unusual problem with my non-denaturing 10% acrylamide gels. After letting the gel solidify for 15-20 minutes, I pull the comb and as always the unsolidified liquid falls into the wells. Up to now this all sounds normal, until I clean the wells by spraying some 1X TBE and realize that that liquid that filled the wells actually solidified and partially filled the wells!

Now when I go to load my wells, my samples sort of perkilate down through the abberant intra-well gel and totally disrupts my banding.

Why is this unsolidified liquid solidifying as soon as I pull the comb? It doesn't even give me time to wash out the wells!

Help!

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[SomeDoc]

It sounds like your gel is not completely solidified when you are pulling the comb out. Cool the gel for longer- 25 min should be sufficient. Alternatively, you can try using a higher percentage concentration for your gel if it is still not solidifying well. When the gel is ready, a helpful hint is to very gently and slowly remove the comb from the cooled/ready gel while it is in the TBE of the electrophoresis unit and thus the wells are hydrated and properly lubricated. This prevents the bottoms of the wells from ripping out.

 

[GreenShirt]

Do as suggested above and also try pouring the TBE in before you gently wiggle the combs out....this'll prevent the wells from collapsing.

 

[ninjapenguin]

I would also try increasing the amount of amonium persulfate and TEMED (maybe 1.5X to 2X of both since they are in such small quantities). That should help the gel polymerize and solidify more.

 

[NYyanx28]

Great ideas. I think before I start changing reagents, I'll try to fill the chamber up with TBE before taking the comb out, this will allow TBE to flood the wells before the unpolymerized gel gets a chance. Thanks!

 

[GradTX]

How old are your APS and temed? I have found that for non-denaturing DNA gels, that the age of the reagents seems to matter much more than denaturing protein gels (like for Westerns). Heck, I would even have to replace my 40% acrylamide before I could use it all.

Sorry if this last part seems crazy OCD. After I pull the comb out, I immediately fill the wells with ddH20 and then suck everything out of them with a gel loading tip hooked up to an aspirator. This seems to remove all that clear gunk that prevents your sample from going to the bottom of the well.

Good luck getting things to work. Science can be stupid like this sometimes.

 

[jackfibi]

How old are your APS and temed? I have found that for non-denaturing DNA gels, that the age of the reagents seems to matter much more than denaturing protein gels (like for Westerns). Heck, I would even have to replace my 40% acrylamide before I could use it all.

Sorry if this last part seems crazy OCD. After I pull the comb out, I immediately fill the wells with ddH20 and then suck everything out of them with a gel loading tip hooked up to an aspirator. This seems to remove all that clear gunk that prevents your sample from going to the bottom of the well.

Good luck getting things to work. Science can be stupid like this sometimes.

Or, you could just buy some pre-cast gels from Biorad (~12$). I did a side by side experiment comparing these gels to other pre-cast gels and my own, and the Biorad gels not only required less protein for detection but the bands ran more even than all other gels.

 

[167649]

Or, you could just buy some pre-cast gels from Biorad (~12$). I did a side by side experiment comparing these gels to other pre-cast gels and my own, and the Biorad gels not only required less protein for detection but the bands ran more even than all other gels.

I used to buy all my gels from BR. When you add it all up, the saved time and anxiety was well worth the price. It's just that much less you have to worry about.

 

[ecoli]

I cool the liquid gel on ice a bit before casting. It quickens the cooling time.

 

[achamess]

I pull the comb out under running ddH20. I find this is sufficient to prevent any unsolidified gell from entering. Doing otherwise leads to aberrant wells.

 

[guru1]

Running tap water is the best what worked for me too.
Also, for westerns, 5% stacker is sooo much better than 4%...

 

[StIGMA]

Not to be obvious, but make sure your comb is the correct size. If so, make sure it is not being pushed forward or backward by the spacers while polymerizing, which could leave space against the glass plates for residue to form. Also, increasing TEMED 50-200% has been successful for me.