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.
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-