HELP making 6X SDS Loading Buffer for SDS-PAGESDS buffer PAGE 6X SB
Posted 04 February 2013 - 08:44 AM
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
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!)
Posted 04 February 2013 - 11:40 AM
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
Posted 08 February 2013 - 05:19 AM
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).
Is it necessary to use 6X dye only
genius does what it must
i do what i get paid to do