How To Make 2M Bisulfite Solution? - Bisulfite Sequencing (Sep/01/2005 )

Hi,
I would very much appreciate your help in making 2M Bisulfite stock solution. The sodium bisulfite granules refused to dissolve even when I tried to bring the pH to about pH 5 with 10M NaOH. Heating did not seem to help.
Thanks,
Anthony

-anthony-

Hello Anthony,

I make my bisulphite solution with a higher molarity and I think it is actually a matter of persistence It won't disssolve totally before you add the NaOH, but you can try to heat it at 50ºC or vortex it a little bit. Anyway, it will always take more than 5 min to dissolve completely even with continuous shaking.
Hope this helps.

Cris

-mCris-

Hi Anthony,

Check this protocol and pay attention to Note 1 in the middle of the page.

http://www.protocol-online.org/prot/Detailed/3160.html

BTW, you should not make stock solution for sodium bisulfite which should be prepared freshly every time before use.

-pcrman-

also, it doesnt hurt to have a few granules remaining, this means you have a super saturated solution of sodium bisulfite, i have done this routinely with no adverse effects on the modifcation reaction.

pcrman is right, make you bisulfite solution fresh for each modification reaction and ensure you do not shake the solution to try and dissolve it, excess oxygen in the solution reacts with bisulfite.

Good luck! Nick.

-methylnick-

Hello Nick!

Normally I shake my bisulphite solution in order to dissolve it but reading your post I realized that maybe I shouldn't do it. So, what could happen if you shake it, can you get incomplete modification of unmethylated cytosines because of this?

Thanks for the help
Cris

-mCris-

I have not witnessed it in my experience, but from what I was told, excess oxygen in the solution reacts with the bisulfite, inactivating it's ability to sulfonate unmethylated cytosines.

Gentle rocking of the tube is fine to dissolve the powder into solution. again, it's fine that you still have granules in solution as this is indicative of a supersaturated solution.

Nick

-methylnick-

Thank you all for your fast and helpful responses. Anthony

-anthony-