Hi all,
I am trying to concentrate a DNA sample using dialysis against 50% PEG, through a 10K cut-off membrane. I made a 50% solution of PEG20K supplemented with 10mM Tris (target pH=7.8), and accidentally found out the pH of that was 3! It took *a lot* of 10M NaOH to bring the pH of the PEG solution back to 7.8 (final Na+ is now at around 250 mM). I can sure dialyse the salt out, but I am now concerned that the low pH was due to PEG oxidation products which I definitely do not want in my DNA solutions. Is freshly ordered PEG powder likely to have a more reasonable pH when dissolved? Should I store PEG under N2? Should I dialyse the PEG solution first, to get rid of the small molecules? This is getting quite complicated... This PEG idea was to avoid centrifuge filtration to concentrate my DNA, as I was having issues with DNA loss...
This document on PEG stability may provide some perspective on your problem:
https://hamptonresearch.com/documents/growth_101/27.pdf
This document on PEG stability may provide some perspective on your problem:
https://hamptonresearch.com/documents/growth_101/27.pdf
Many thanks, Adam! The document was quite eye-opening! The conductivity part was particularly worrisome. Interestingly, some Mws were more stable than others. The jar of PEG I had I borrowed at a colleague's lab. So I will probably get a new batch, and store the powder at -80 in smaller aliquots.
There is more to it, it's not just regular DNA, these are nano-structures. I do not know how they will behave in EtOH,, and I do not want to dry them. I know they behave OK in PEG, so I might precipitate in PEG instead.
OK - that makes sense.
If you are worried about contamination from the PEG you can apply a boat load of dry PEG to the outside of your dialysis bag, I've done this for proteins in the past as a quick concentration step and didn't notice a pH change. I get a kitty litter tray and just pile the PEG over my dialysis bag, keep an eye on it to monitor the sample volume.
Thank you, Neville! The dry PEG trick sounds like something I should try.
Did it work Andrey? I just tried your Cloud-point PEG Glass Surfaces protocol from bio-protocol.org, works like a charm!
Hi Neville -- yes, it worked, mnay thanks! I am glad you liked the PEG protocol. An additional 'tweak' we implemented in the last few months is to sonicate at every step involving surfaces -- APTES, PEG, and NH2-quencher.
Hi Andrey,
Glad it worked.
Could you clarify how long you sonicate for for these steps? In the protocol I have the acetone washes are brief (10s or so). Are you doing an extended sonication in the acetone, and also how long do you sonicate in water after the PEG and NH2-quencher? Thanks!
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