Cloudy Alkaline Phosphatase Buffer! - my AP buffer gets cloudy after autoclave (Jun/08/2008 )
Hello all,
when I make Alkaline phosphatase buffer ( for visualization of my membrane bands to mix with NBT/BCIP) and after autoclaving I get a cloudy solution with some debris floating...I don't know what they are.
I use the method of Sambrook, and it seems to be happening to my other friends too...I even filtered the solution before autoclaving but still I get cloudy solution? I tested other Tris, NaCl and Mgcl2 from other labs, but I still face the same problem.
does this happen to you too? how to overcome the problem?
when I make Alkaline phosphatase buffer ( for visualization of my membrane bands to mix with NBT/BCIP) and after autoclaving I get a cloudy solution with some debris floating...I don't know what they are.
I use the method of Sambrook, and it seems to be happening to my other friends too...I even filtered the solution before autoclaving but still I get cloudy solution? I tested other Tris, NaCl and Mgcl2 from other labs, but I still face the same problem.
does this happen to you too? how to overcome the problem?
May be some of these recipes have your answer:
http://search.vadlo.com/b/q?sn=158621799&a...uffer&rel=1
..
why do you autoclave? i never did and had no problems with the buffer.
if you feel the need to sterilize then use filters.
if you feel the need to sterilize then use filters.
autoclave is mentioned in various protocols. I made the AP buffer again, this time I have crystals, must be MgCl2, then I autoclaved and filtered with whatman, it seems ok this time.
if you feel the need to sterilize then use filters.
autoclave is mentioned in various protocols. I made the AP buffer again, this time I have crystals, must be MgCl2, then I autoclaved and filtered with whatman, it seems ok this time.
If you filter significant amount of crystals, you change the buffer composition. But I am sure you have thought about it.
if you feel the need to sterilize then use filters.
autoclave is mentioned in various protocols. I made the AP buffer again, this time I have crystals, must be MgCl2, then I autoclaved and filtered with whatman, it seems ok this time.
If you filter significant amount of crystals, you change the buffer composition. But I am sure you have thought about it.
yep, that's right. we don't use much MgCl2 in the composition, and if we filter it we might lose it.
NaCl can easily dissolve, and Tris also never makes cloudy solution, so it must be MgCl2....I'll try to run a western soon, and will update you with the outcome.
Thanks.
MgCl2 is pretty soluble too; 54 grams per 100ml... in fact more soluble than NaCl (36g/100ml). Maybe your autoclave (or water) is dirty and is introducing something to the solution that is causing a precipitate.