how much of each loading dye component - (Sep/01/2005 )
I just want to ask how much in milliliters of each component am i going to prepare for this 6x loading dye:
0.25% Bromophenol blue, 0.25%Xylene cyanol FF, 15% Ficoll Type 4000, 120 mM EDTA
when it says '0.25%' or so of something in a recepy it is normally meant as w/v (= weight per volume), if the meantioned substances are solid. For example 100% (w/v) would mean dissolving 1 kg of the substance in an end volume of 1 liter. For the EDTA you should prepare a higher concentrated stock solution.
For 100 ml of your loading dye this would mean:
0.25 g bromophenol blue sodium salt
0.25 g xylene xyanol FF
15 g Ficoll Type 4000
24 ml of a 0.5 M EDTA solution pH 7.6- 8.0 (EDTA won't dissolve at lower pH)
fill up to 100 ml with distilled water.
I hope I made no mayor mistakes in calculating through this, but I hope this will help you anyway.
The amount of the dyes is unimportant, except that you be able to see them on your gel. To additionally confuse you, I recommend switching from Xylene cyanole and bromphenol blue to a single dye, orange G, which runs ahead of all of the DNA bands, and does not interfere with imaging the bands with a camera.
That seem like a very high EDTA concentration. With the concentration that high, it really might matter that you use this at a 6x dilution, rather than a higher concentration (which I often do, to avoid adding water to the DNA). We've never added EDTA to our loading dye, although I can see that it might be a good idea.
I might just add that in our lab we're using a loading dye I make myself out of 30% sucrose with 0.3% Orange G as a dye . It works fine, though because of the sucrose it has to be filter sterilized.