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Inoue Methode - How to make the Buffer for the Inou eMethode (Feb/18/2006 )

Hi,
I wanted to make some "ultra" competent cells using the Inoue-Method, which is discribed in Maniatis
protocol 24. My problem is that therefor a buffer is needed with contains PIPES, but I don't get that substance solved. I raised the pH to 7.5 but all what happened was, that the buffer turned brown, but still the PIPES does not get into solution. So when I want to filter the buffer all stays in the filter. What can I do? Has someboady used this buffer and can help me?

-TMentzel-

hi
you can dilute with gentle heating (up to 50°) and let swirl overnight... but don't forget to cover it with alminium paper in order to minimize evaporation. and if possible, do all under fume hood.

-fred_33-

QUOTE (fred_33 @ Feb 20 2006, 05:09 PM)
hi
you can dilute with gentle heating (up to 50°) and let swirl overnight... but don't forget to cover it with alminium paper in order to minimize evaporation. and if possible, do all under fume hood.


Well, I have done this. I diluted to the concentraction of the final buffer (50mM) and heated (most likely a littlebit more than 50° but not much more), but the PIPES didn't go into solutiuon. So I switched of the heat and swirled it over night, but it didn't help at all.

-TMentzel-

maybe you can ask to a company which sold you the PIPES?... or any of one

-fred_33-