7 replies to this topic

[Prep!]

Might sound like a stupid question, but wanted to ask if we can vortex a 30% acrylamide solution without doing any hard to it??
Might be too silly a question so could not get anything on the net!!! lol!!!
I have to do it as do run a denaturing IEF i have to add urea and urea dissolves easily in acrylamide water mix than only water!!!

[Prep!]

Pradeep Iyer, on Oct 26 2009, 09:27 AM, said:

Might sound like a stupid question, but wanted to ask if we can vortex a 30% acrylamide solution without doing any hard to it??

I meant  harm (not hard!!!)

[Prep!]

sorry folks for lengthening the querry... but just wanted to inform that i asked the querry as i m not getting repeatable gels in terms if isoform resolution that is why this doubt creeped up my mind!!!

[mdfenko]

vortexing will not harm the molecules but you may be increasing the amount of dissolved oxygen. oxygen will interfere with polymerization.

after vortexing you should degas (before adding persulfate).

[Prep!]

mdfenko, on Oct 26 2009, 08:37 PM, said:

vortexing will not harm the molecules but you may be increasing the amount of dissolved oxygen. oxygen will interfere with polymerization.

after vortexing you should degas (before adding persulfate).

point taken!!!  

[miBunny]

I'll second mdfenko, dissolved oxygen in the solution will screw up the polymerization.  If anyone is interested, APS and TEMED react together to form a free radical that initiates polymerization.  Oxygen quenches the free radical stopping polymerization-  My college biochem prof drilled this reaction into our heads!

[Prep!]

miBunny, on Oct 28 2009, 07:59 AM, said:

I'll second mdfenko, dissolved oxygen in the solution will screw up the polymerization.  If anyone is interested, APS and TEMED react together to form a free radical that initiates polymerization.  Oxygen quenches the free radical stopping polymerization-  My college biochem prof drilled this reaction into our heads!

yup me aware of that.. that is the reason we overlay the seperating gel with saturated butanol!!!
thanx anyways!!!

[mdfenko]

Pradeep Iyer, on Oct 27 2009, 10:57 PM, said:

yup me aware of that.. that is the reason we overlay the separating gel with saturated butanol!!!

the overlay is to ensure a flat interface. over the years i have overlaid with water, buffer, butanol and isobutanol.

butanol and isobutanol won't mix with the aqueous acrylamide solution so can be applied with a little less care than a miscible overlay.

never worried about introduction of oxygen at this stage.