HELP making 6X SDS Loading Buffer for SDS-PAGE - (Feb/04/2013 )

Hello,

I need some tips on how to make a 6X SDS loading buffer for use in SDS PAGE.
My biggest problem is getting the SDS into solution.
Last time I made it, I heated the solution on the lowest possible setting on our stir/heating plate to get it into solution. Once the SDS appeared to be dissolved, I turned the heat off and added the DTT and bromophenol blue. While I was aliquotting the solution for storage, it almost seemed to be becoming more solid. Indeed, I just removed an aliquot from our -20C and it is impossible to pipette and seems to be more of a gel-like solid than a liquid. (Our 6X loading buffer has always been thick in the past, but not this thick). I also have tried making the buffer without heating at all, and the solution becomes super thick even quicker.

This is the recipe we use for 10mL:

3.75mL 1M Tris pH 6.8
1.2g SDS (solid)
6mL glycerol (100% stock)
0.006g bromophenol blue
0.462g DTT

Any help would be greatly appreciated, or if anyone has a protocol for making a 6X SDS loading buffer that works for them I would also appreciate that! (Please tell me how you get the SDS to dissolve without turning the solution into a solid!)

12% SDS should be soluble at 37 degrees pretty easily, though I don't know how the addition of glycerol would affect this.

My 6x recipe (20 ml) is:
616 mg DTT
154 ul 20% SDS
969 ul 1M TRIS pH 6.8
8 ml (10.08g) GLYCEROL
800 ul 2% BROMOPHENOL BLUE IN ETOH
ADD H2O UP TO 20 ml
ALIQUOT INTO 1 ml AMOUNTS AND FREEZE AT -20

Hi

Is it necessary to use 6X dye only

shrenik3004 on Thu Feb 7 23:38:22 2013 said:

no. you can make 2x, 5x, 10x, 12x,... your limitation is the solubility of the components (we keep some components separate so that some parts are higher concentration than others).