Preparing Gel Loading Buffer - (Jun/28/2007 )

Hello All,

I could really use some help. I'm making 6X Gel Loading Buffer type III. The recipe is .25% bromophenol blue, .25% xylenecyanol FF and 30% glycerol in water. So I added .25g of both dyes to 3ml of glycerol and qns up to 10mls with water. Problem is my loading buffer looks green in the tube. When I spilled it all over my hands the solution appeared blue. Could the pH of the water cause the solution to look more green? Anyone else had this problem?

Thanks

first, you made your solution 2.5% bpb and xylene cyanol.

second, xylene cyanol will look greenish.

dilute the solution with 30% glycerol (to 100 ml) and all should be well.

we also use bpb

Don't worry about the green color, it is as expected. What you described is perfectly normal. The green is due to xylene cyanol. After you prepare the buffer, it would have greenish color, but when you mix it with sample it will turn blue, so it is also one way to recognize whether you has added anything into your loading buffer or not (the color is likely due to pH, since I have added just elution buffer with the loading buffer and it changed color all the same).

The loading buffer with both xylene and bpb will give you two bands of dye when you run gel: 1 green band that runs slower (at the range of about a few kb on 1% agarose gel), and 1 blue band that runs faster (at about <500bp if I am not wrong). Some people use only bpb, in that case there should be only one blue band in running gel.

Those dyes are both pH indicators, so as Almasy said, the colour will change when you add DNA. Or just make the dye in TE instead of water.

Thank you for the input. I ran some on a gel and it looked fine. I appreciate all the responses.