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polyacrylamide gel problem....slipping

EMSA polyacrylamide gel problem

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5 replies to this topic

#1 stelane

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Posted 28 September 2011 - 07:06 AM

Currently having a problem where my polyacrylamide gels slip out between the glass plates they have polymerized in.

I am using a 5% gel in Tris-Gly buffer, the gel is made using the same buffer. Sometimes, not every time though, when the buffer is added to the gel box the gel will run slowly out through the bottom, adding current will speed this process up. The polymerization occurs OK, I always leave some over in a beaker to make sure. I don't know why this happens, it is random but very annoying and is resulting in a lot of wasted time. The glass plates, spacers and wells are cleaned and dried thoroughly before each use. Any help would be much appreciated.

thanks,
Steve

#2 mdfenko

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Posted 28 September 2011 - 07:33 AM

you may have residual soap (or detergent) on the plates

and/or

your plate(s) may be warped
talent does what it can
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#3 stelane

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Posted 28 September 2011 - 09:48 AM

Thanks for the answer, don't think it is residual detergent though, I scrub the plates clean, rinse thoroughly under tap water and then rinse three times in RO water. But warped plates is interesting, I was wondering if clamping the plates together may cause the plates to slightly bow out in the centre sometimes and bow in at other times?

#4 mdfenko

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Posted 28 September 2011 - 10:13 AM

if you are talking about when you are pouring the gel then you must set the clamp directly on the spacer.

if you clamp inside the spacers then you will close the gap between the plates. when you release the clamp the plates will separate and you will have a gap between the plate and the gel.
talent does what it can
genius does what it must
i do what i get paid to do

#5 stelane

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Posted 28 September 2011 - 10:29 AM

Thanks for the reply.

Yes I was talking about when I pour the gel and no I am clamping onto the spacers themselves, but was wondering if the force exerted by the clamps onto those spacers could potentially cause bowing or warping of the glass. I've just been told that the clamps we are using were originally for very large gels whereas the ones I'm making are about 160mm x 180mm, so wondered if there might be an issue there i.e. strong clamping pressure on small glass plates? I've just gone out and bought some new clamps that exert less pressure maybe this will help maybe not!

#6 BioMiha

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Posted 29 September 2011 - 12:25 AM

You just have to be really careful to align the front and back plates perfectly so the rubbery bit closes them tightly while the gel is solidifying. Also if the glass is chipped at places that could be the reason.





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