Jack D. Pollard, Jr.
These denaturing dyes are used on urea-based polyacrylamidesgel for the separation of single stranded nucleic acids. They are basedon loading dyes used in the Herschalg and Cech kinetics papers.
Make the loading buffer in 1 liter amounts without dyes,and then aliquot 10 ml fractions into 15 ml Falcon tubes and store at 4°Cin the fridge located by the bench 15. This stock solution can then beused to make many dye containing variants.
Prepare a loading buffer stock as follows:
to make 1 liter of
2 mM Tris, pH 7.5
20 mM EDTA
mix the following:
480 g of urea
40 ml of 0.5 M EDTA
2 ml of 1M Tris pH 7.5
Dilute to 1 liter with house distilled water, and usemagnetic stirring over to several hours to ensure that the urea dissolves.Check the pH, but it is usually fine. Filter this urea loading dye stockthough and 0.2 micron filter to remove any debris or bacterial contamination.
Prepare stocks of xylene cyanol and bromophenol blue asfollows:
Dissolve 700 mg of bromophenol blue in 5 ml of urea loadingbuffer.
Dissolve 700 mg of xylene cyanol in 5 ml of urea loadingbuffer.
To achieve dye concentrations such that 1 mlis visible on a sequencing gel, add 0.6 ml xylene cyanol stock solutionto 50 ml of loading buffer.
To achieve dye concentrations such that 1 mlis visible on a sequencing gel, add 0.2 ml bromophenol blue stock solutionto 50 ml of loading buffer.
To achieve dye concentrations such that 1 mlis visible on a sequencing gel, add 0.2 ml bromophenol blue and 0.6 mlxylene cyanol stock solutions to 50 ml of loading buffer.