3 replies to this topic

[SPombe3]

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!)

[bob1]

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

[shrenik3004]

Hi

Is it necessary to use 6X dye only

[mdfenko]

shrenik3004, on 07 February 2013 - 03:38 PM, said:

Hi

Is it necessary to use 6X dye only

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).