I am having trouble with my tris-glycine running buffer. It's very cloudy, I would guess because the SDS has come out of solution. I don't remember having this problem in the past, and I have remade it a couple times and it is still cloudy. I can heat it and it will go back into solution, but not for long.
Any ideas what would be causing this? Is it something I should be worrying about?
Here is the protocol I used to make 1L 10x Tris-glycine buffer (the 10x and 1x are both cloudy):
30g Tris Base
144g Glycine
10g SDS
Thanks.
per-warm-up your buffer at 40-50C water bath, remix and dissolve well before use if you already mix the SDS all together. usually SDS made separately a stock, warmup o dissolve before mix with the buffer other ingredients. You should be fine then. Good luck and best regards o your research. xw
per-warm-up your buffer at 40-50C water bath, remix and dissolve well before use if you already mix the SDS all together. usually SDS made separately a stock, warmup o dissolve before mix with the buffer other ingredients. You should be fine then. Good luck and best regards o your research. xw
Hi Anna, it could be the lab temperature, or maybe some contaminants in the water or the recipient. You should check your reactives quality. This is the recipe we use and works fine, it has more glycine :) http://cshprotocols.cshlp.org/content/2006/1/pdb.rec9132
Thanks for the help! The Tris base is new, but I checked my calculations and everything was as it should be. I then checked the pH and it was low (7.9, should be 8.3). I am not sure what I can do about this, because I have read that you should not adjust the pH of the running buffer.
I will try the protocol you recommended Maria, maybe using the 10% SDS solution will help.
Please use this protocol-Tris-30.2 g, Glycine-188 g, 100 ml of 10 % SDS and make the volume to 1L. Use milli Q/ RO water, it will definitely work. Gd Luck.
We also had trouble with solubility when making 10x, we think because of glycine solubility (max 250g/L). We used 188 g glycine for 10x. Water bath helped. But I would recommend making 5x instead.
Hello, hope you solved your problem. We have the similar situation with 1X transfer buffer. It's getting cloudy after using one time. We made 10X transfer buffer as below: Tris-58g; glycine-29g; SDS-3.7g, dissolve in 800ml DW (milli Q/ RO water), then diluted to 1X transfer buffer and add 200ml of methanol. The 1X transfer buffer's even getting more cloudy when we store at 4 degree.
Is there anybody who encountered the same problem? Can you suggest me any of your opinion? Thank you.
Tu
Hello, I have already used the same formula and mixed using magnetic stirrer for 10 minutes at room temperature. It works out.
I am having the same problem. The buffer is getting cloudy after one use and all the proteins are stucked up near the interface of stacking and separating gel. Please help!
I had the same problem with dissolving of sds. I changed the quality, using more pure sds and the problem was solved.
10X Tris-Gly-SDS is definitely soluble. The problem may be your SDS, especially if the stock is old and/or has been not stored dry. SDS tends to hydrolyze to form dodecanol which is not soluble and will make SDS solutions acidic. If enough is present, solutions will apear cloudy. Make your running and sample buffers with freshly purchased electrophoresis-grade SDS.
Dear all,
1. pH of my running buffer is more than 8.3 (ph 9.0). Since we should not adjust the ph of the running buffer should I add glycine to make it slightly acidic . Based on the reply from Sir Oliver Krohn
, Tris base can be add until the pH reaches 8.3.
2. My lab don’t have an access to the milliQ water(due to faulty machine) . Is it okay for me to just use distilled water instead of MiiliQ/Deionized water to prepare all the buffer related to the SDS-PAGE
Thanks,
Make 10X tris-glycine in 900ml and SDS in 100ml MQ separately, then add them together to avoid frothing.
Most importantly Store at room temperature, if you store at 4 C It will become cloudy due to SDS.
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