a big mystery - failure of gelling (Apr/07/2007 )

hi guys,

we're having a problem with gel solidification, something still wrong with the stacking gel though we prepared and adjusted chemicals freshly and for many times..i thought the problem was with APS or TEMED, but neither was the reason...also, bubbles appeared each time though we degas the solution before applying into the system..
what could be the cause(s)...i thought that lab conditions (cold air maybe) may play a role here..

welcoming your recommendations

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APS and TEMED have been fresh but what about acrylamide and bisacrylamide?

isn´t it true that the polymerization reaction needs O2? so may try w/o degasing

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O2 suppresses polymerization. Dont mix after degas. Add a layer of water-saturated 1-butanol on top of your gel layer to insulate O2.

Sometime, the whole bottle of APS is no good because water in the air decomposed it and you need to get a fresh one.

Bubble would suggest either the temperature of the gel solution was low, or you added too fast to the gel case. it needs to be warmed to rt. You need to remove these bubbles by tapping the glass plate. Try to add the gel solution from one side of teh gel case.

If you are not using ready-to-use gel mix solution, check if you had Bis in the reaction. Without Bis, you will get thick but still moving liquid, not a gel.

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Hi! Strawberry

I hope you find some answers on your mystery here :
http://www.biocompare.com/technicalarticle...om-Bio-Rad.html

Some start up suggestions:

Prepare fresh PSA and TEMED

To increase rate of polymerization - 37C work well. But it is not well for further resolution of your gel ( slow polymerisation - more regulary formation of pores in gel)

Check youe acrylamide stock! We use Amberlite MB 150 resin to remove acrylic acid and other inorganic impurities which can have negative for polymerization. Then we filtrate on 0.45um. Sometimes we keep stock with resin. If mystery will return, for final prepare your new acrylamide stock

GOOD LUCK!

Reply to the Bearer

O2 play a role in riboflavin photochemical polymerisation

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thanks all for your suggestions
thanx circlepoint for the link

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Following Genehunter-1 reply: Excess of Oxygen inhibit polymerization ( inhibit elongation of polymerization process because of quench of free radicals generating by PSA) .

http://nationaldiagnostics.com/article_inf...p/articles_id/6

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If your stock solution is in good quality (meaning getting from the supplier instead of making it), there is no need for degassing.

Another thing, when did you put your SDS solution? before or after degassing?

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You may also check the APS. It needs to be fresh and kept away from light. The pH of the buffer you use is important too. Make sure all your solutions are made properly

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Hi Timjim! If your stock solution is in good quality you still need degassing because to prepare work solution you also add water and tris which contain oxygen too. SDS should add after degassing otherwise big foam formation will occure

I don't recommend only to degasse 20% work solution to prepare gradient gels because of possibility of polymerization in gradient maker!

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