No polymerization...at all.2 batches.

cytokine

I've read the prior posts re these problems. The only difference is that we're mixing our own acrylamide (sigma a9099-100g) and bis (sigma m7279-100g) and making our own 16% pfa solution. The va044 is the wako one.
Following protocol...Degas and nitrogen flush. Tight cap. Incubated at 37...and nothing. Made second batch changed pfa ph to 7.45 after reading posts recommending this. Nothing.
Took first batch out overnight from freezer and left at room temp in conicals planning to discard. No polymers.
Left first and second batch in single conical at 38 water bath overnight. Nothing.
So I wonder what next? Should we stick with commercial solutions. Are folks mixing their own? What should we look out for? Any advice is immensely appreciated. Thank you.

cytokine

So I'm wondering. Should we be checking the final hydrogel solution ph? If so what should it be? Anything else? Thanks again.

Clara

Hi, I used homemade solutions and in my first attempt I had problems because the pH of my PFA stock wasn't well adjusted. Once I corrected that, my gel polymerized every time. I use PFA from Merck (8.18715.0100), Acrylamide also from Merck (1.10784.1000) and bis from Sigma (M7256) and I dont degas, I use mineral oil instead. I dont think your problem is that you make your own solutions, maybe you are missing some little detail, something in the recipe maybe. Is your PBS pH correct? I use 10X PBS pH 7,4. Sorry! I know I 'm not beeing very helpfull... keep trying, I'm sure you will find out what the problem is.

cytokine

Thanks. We're going to try another batch. I'll check re PBS ph. Should the final mix be ph adjusted?

Adam

If the PFA has been pH'd, then (in my experience) the hydrogel should be fine.

Just to check, what percentages of acrylamide, bis and PFA do you have in the final solution? If you've reduced these much from the original 4%, 0.05% and 4% from the original paper, your hydrogel will only polymerise to a solid inside the tissue, and not in the rest of the solution.

If you are using a high density hydrogel, then I'd check the actual temp of your water bath.

cytokine

Thanks Adam. We made a 1% mix. I thought that would still have some polymerization but if not then maybe we're good.

Adam

If it's 1%, but you've pH'd the PFA, and degassed the tubes before polymerisation, then the solution should polymerise within the tissue, but only thicken very slightly outside of it. I'd start clearing your samples to check.

cytokine

Great. We also made a 4%today and it polymerised in both the nitrogen and oil covered tubes. Thank you all.