melting a polyacrylamide gel? urgent - (Feb/20/2007 )
Has anyone been able to or knows how to melt a polyacrylamide gel? This gel has been put inside a slicia chip. It went through gel polymerization using UV light. However, when DNA is concentrated on it, the gel gets permeable and DNA goes into the gel rather than collect on the outside bank of the gel (which is the purpose for this gel). I would like to reuse this chip again where I wash out this old, bad gel, and reform a new gel polymerized gel using stronger concentrations of the same reagants.
Does anyone have any suggestions or tips on how to "de-polymerize" this old gel on the chip without hurting the chip itself? Are there chemicals that can be used to flush through the gel area that will weaken the crosslinking that formed during UV?
peroxide and perchloric acid and warm-to-hot water bath will dissolve polyacrylamide.
Do you mean hydrogen peroxide?
Your reply leans towards using a strong acid to weaken the gel crosslinkers along with applying warmth to the chip in some way (in this case, a hot water bath). Am I interpreting this correctly?
Also, would placing the chip under a hot UV lamp also melt the gel inside the silica chip? Or would it just melt the chip material?
Your reply leans towards using a strong acid to weaken the gel crosslinkers along with applying warmth to the chip in some way (in this case, a hot water bath). Am I interpreting this correctly?
Also, would placing the chip under a hot UV lamp also melt the gel inside the silica chip? Or would it just melt the chip material?
yes, i meant hydrogen peroxide in combination with perchloric acid.
you are correct about applying warmth.
i never tried uv (or hot uv) to dissolve polyacrylamide nor do i know the effect of uv on silicon chips so i can't knowledgably answer your question.
i do know that the perchlorate/peroxide/heat mix works and i don't think it will affect the silicon (other components on the chip, you may have a better idea).
if you want, i can get the procedure for you.
you are correct about applying warmth.
i never tried uv (or hot uv) to dissolve polyacrylamide nor do i know the effect of uv on silicon chips so i can't knowledgably answer your question.
i do know that the perchlorate/peroxide/heat mix works and i don't think it will affect the silicon (other components on the chip, you may have a better idea).
if you want, i can get the procedure for you.
Yes, I'd really like to see this procedure even if I only extract a part of it for my own use. Also, would you know the chemical reaction that takes place when hydrogen peroxide reacts with polyacrylamide, as in which bonds are attacked chemically? Thanks.
I am afraid that the condition would be too harsh and that your samples will be lost/damaged as well. I would suggest you to incorporate disulfide bond containing cross-linker to make polyacrylamide gel so you can release DNA by using mild condition in the presence of a reducing agent. Biorad sells this cross-linker and you can substitute the bisacrylamide with it. The % of it depends on the size of the DNA that you are trying to entrap or separate.
wet oxidation method for solubilizing gelled acrylamide.
1: prepare fresh: 60% perchloric acid and 30% hydrogen peroxide
2: place gel slice in a sealable (teflon or polyethylene coated cap) glass vial
3: add 2 volumes peroxide (slow, dropwise) then add 1 volume perchloric acid (slow, dropwise). seal the vial
4: incubate in oven at 70C until gel is dissolved (typically, 3 hours)
this method was used to dissolve gel slices for scintillation counting so the rest of the method (neutralizing and addition of scintillation cocktail) will not be necessary. just rinse the chip with distilled or deionized water to remove residual peroxide and perchlorate.
good luck