Lysis buffer - (Feb/25/2010 )

Hi!

I'm trying to prepare 10X lysis buffer, but I have problems with solubility. The buffer should be 0,5 M EDTA; 0,5 M Tris; and 10 % SDS. I adjusted pH just above 8, but there is something that doesn't want to dissolve. Could you please help me!?

It is the SDS I guess. You should "warm" it to allow the SDS to solubilize

i would also guess it to be SDS. warm it

Yes, I tried also to warm it to 70-80 C... It didn't help. There solution is otherwise clear, but there is a thick precipitation at the top...

Piglet on Feb 26 2010, 08:02 AM said:

Why don you try to add the SDS to the final 1X solution? I know that a 1%SDS solution is ok, I never tried such amount of SDS.

Because I need to have 10x buffer to add to my samples... I have liquid samples, and if I add 1X buffer the samples would dilute too much. I think now I'll try to make more concentrated solutions of each ingredient, and combine them as liquids. Before I had just EDTA as a liquid.

If you have never made 0.5 M EDTA solution before, you should know that it won't go into solution at all until neutralized with NaOH pellets. Also, you can't make 1 M EDTA -- it is not sufficiently soluble even when neutralized.

Your life would be much simpler if you made a 5x buffer, using 0.5 M EDTA stock solution, 1 M Tris solution, and 20% SDS.

But why should life be simple and without challenges

Thank you all for your comments, finally after a week in + 70 C my buffer seems to be clear!

10% SDS should be fine at room temp - it will ppt at about 18 deg C though. I suspect it is the EDTA precipitating, like it does in TBE, as it is near its solubility limits, the addition of ions or slight changes in pH will cause it to come out of solution.

Piglet on Mar 1 2010, 06:29 AM said:

I also suspect it is EDTA. A week in 70C.. crazy haha.
however if at all time u could keep it simple, please do. Research is a race against time.