I'm making a stock of O'Farrell solution for SDS-PAGE gel casting. The recipe calls for 1.5M Tris pH 8.8 and 0.4% SDS in water, final pH 8.8. Each time I or a lab-mate try making this solution, we see a cloudy precipitate/swirl (the SDS?) appear that does not disappear with changing pH or temperature.
We've tried making the solution using stock 20% SDS (pH 7.2) and stock 2M Tris (pH 8.8), as well as using the powdered form of each with the same result each time.
(Both stock solutions are clear and this is being done at room temperature; we've used the same recipe before, and it worked then....)
Thanks!
The Tris is just almost 3 years old and has been open for about 8 months. So according to the company, it should be fine (and they said they had no other complaints about the lot number). I guess something got in the bucket...oh well :( . Glad to have finally figured it out though.
Thanks, all, for the help/advice!
In SDS PAGE gel we use 0.1% SDS, Stock prepared is 10% in water. We observed precipitates when we store stock below 4 degree celsius.
Thank you Vinod Vijayan for your advice, that was by mistake we kept it in refrigerator along with all SDS PAGE reagents and we could see it precipitated.
If the room temperature goes below 10, you may usually experience this esp. in winter. just keep the bottle in 55C oven or water bath for 5 mim and then use it....
Our SDS is at room temp (around 22*C), and we make the SDS-Tris buffer at room temperature as well. so I don't think low temperature is the problem. Introducing heat (via a stirring hot plate) did not result in the SDS-Tris buffer solubalizing. This morning I tried pouring an SDS-PAGE gel with this buffer to see if it might work; however, the samples did not separate properly (as compared to hundreds of previous attempts).
And using new SDS and new Tris didn't make a difference...is it possible that the pH meter (which we clean with water before using) is affecting the SDS?
I'm not sure exactly what was going on, but apparently it was a problem with my Tris; using another lab's Tris fixed the problem (changing the SDS didn't make a difference).
I always add the SDS to the Tris (both the 20% SDS and 2M Tris pH 8.8 have no problem going into/staying in their respective solutions), and a single drop of SDS causes the precipitate/swirl to form (it looks kind of like a crystal ball, the way the precipitate/crystals swirl). I'm thinking my Tris has gone bad; there's no expiration date on the bucket and the lot number gave an error online, so I'll be calling the company to ask tomorrow....
The Tris is just almost 3 years old and has been open for about 8 months. So according to the company, it should be fine (and they said they had no other complaints about the lot number). I guess something got in the bucket...oh well :( . Glad to have finally figured it out though.
Thanks, all, for the help/advice!
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