How to prepare LiCl washing liquid - (Aug/16/2010 )
I have another question. The ChIP buffer and reagent are home-brewed according to the ChIP Kit description (Millipore). The problem comes from LiCl washing solution (10mMTis-Cl pH 8.0, 1mM EDTA, 0.25M LiCl, 1%NP-40, 1% sodium deoxycholate). For the first time, I first add sodium deoxycholate, then add NP-40, but I discovered that solution become turbidity and then precipitated; I think this phenomenon may be due to the hygroscopic properties of NP40 which compete with the dissolution of sodium deoxycholate. Thus, for the second time, I join the NP-40, then add sodium deoxycholate, and I got clarified solution which then successfully applied to the test of that day. And I had thought I have solved this problem, but in the next day the precipitation occurred again in reagent bottle. I do not know if anyone here encountered this kind of situation? Someone told me that the reagent inside the KIT is clarity. I had it gone wrong? How can I prepare? Yearn Reply
Hi Biolily,
Did you prepare the wash solution using stock solutions or did you add things like the Deoxycholate and NP-40 directly? If it is the later I would suggest first making up the individual components as stock solutions (e.g. 1M Tris-HCl, 0.5M EDTA, 5M LiCl, 10% NP-40, 10% Sodium Deoxycholate). Also I have heard rumours from various sources that Sodium Deoxycholate powder bought from some companies (don't know which!) have problems going into solution. I use a powder from Sigma-Aldirch which seems to work fine! Personally I have not had any trouble making the buffer, once prepared and stored in the fridge I have not noticed anything coming out of solution.
Hope this helps,
Jon
That lysis buffer generally goes solid when there is no NP-40 in it. Once NP40 is in it - you can safely store it at 4degC without any chunks (this is my experience). How old is your NP-40? Are you using an already-dilted stock of NP40? Be careful as it degrades in solution over time.
Clare
Biolily on Tue Aug 17 04:48:29 2010 said:
I have another question. The ChIP buffer and reagent are home-brewed according to the ChIP Kit description (Millipore). The problem comes from LiCl washing solution (10mMTis-Cl pH 8.0, 1mM EDTA, 0.25M LiCl, 1%NP-40, 1% sodium deoxycholate). For the first time, I first add sodium deoxycholate, then add NP-40, but I discovered that solution become turbidity and then precipitated; I think this phenomenon may be due to the hygroscopic properties of NP40 which compete with the dissolution of sodium deoxycholate. Thus, for the second time, I join the NP-40, then add sodium deoxycholate, and I got clarified solution which then successfully applied to the test of that day. And I had thought I have solved this problem, but in the next day the precipitation occurred again in reagent bottle. I do not know if anyone here encountered this kind of situation? Someone told me that the reagent inside the KIT is clarity. I had it gone wrong? How can I prepare? Yearn Reply