Preparation of 6X DNA Loading Dye (Bromophenol blue and Sucrose) Alternative Method

OVERVIEW

This method uses 40% sucrose solution instead of sucrose powder. This method is convenient and easy, and does not involve adjustment of volume.

REQUIREMENTS

Reagents and solutions
Bromophenol blue
40% (w/v) Sucrose solution (sterile)
Deionized / Milli-Q water

Equipment and disposables
15-ml screw-cap graduated polypropylene centrifuge tube
Centrifuge (for 15 ml tube) 
Tube rotator (optional) 
Vortex mixer (optional)

COMPOSITION

Composition of 6X DNA loading dye 
0.25% (w/v) bromophenol blue
40% (w/v) sucrose

Composition of 1X DNA loading dye 
0.042% (w/v) bromophenol blue
6.67% (w/v) sucrose

OBJECTIVE
Preparation of 10 ml of 6X DNA loading dye containing bromophenol blue, and Sucrose

PREPARATION

To prepare 10 ml of 6X DNA loading dye, weigh out 25 mg bromophenol blue. Transfer it to a 15-mL screw-capped graduated tube. Add 10 ml of 40% sucrose solution. Mix vigorously using a vortex mixer or tube rotator to dissolve bromophenol blue and xylene cyanol FF in 40% sucrose solution. 

Note
This method is simple and convenient as it does not involve readjustment of solution volume. There will not be any significant change in the volume of the 40% sucrose solution when you dissolve small amounts of  bromophenol blue in it.

Tip
We recommend using disposable nuclease nuclease-free (DNase- and RNase-free)15 ml screw-capped graduated tubes instead of beaker as all the procedure can be done in a single tube.

Precaution
If you expect that Bromophenol blue can co-migrate with the DNA fragment of interest, reduce the dye concentration (add 3-5 mg of dye). Bromophenol blue co-migrates with the 300-400 bp DNA fragment and will mask the DNA band.

STORAGE
Store the solution in the freezer (-20°C). Solution can be stored at 2 – 8°C for a few weeks.

Precaution
Sucrose-containing loading dyes are prone to mold-like growth. Avoid keeping it in the refrigerator or room temperature. 

Tip
It is recommended to store the solutions in small aliquots (1 ml) at -20°C.

APPLICATIONS
This solution is used for loading DNA samples onto non-denaturing agarose or polyacrylamide gels.

Follow the table to prepare 6X agarose gel loading dye of various volumes.
Reagents / Volume5 ml10 ml25 ml50 ml100 ml
Bromophenol blue12.5 mg25 mg62.5 mg125 mg250 mg
Xylene cyanol FF12.5 mg25 mg62.5 mg125 mg250 mg
40% Sucrose solution5 ml10 ml25 ml50 ml100 ml

HOW TO USE

  • Take 1 part dye and 5 part DNA sample (e.g., 1μL 6X loading dye and 5μL DNA sample). Mix it and load in the well of agarose gel or polyacrylamide gel.
  • If you need to take a DNA sample less than 5μL, add a water or electrophoresis buffer to bring the final volume to 6 μL.

CALCULATOR

Use Calculator to calculate the amount of different components of 6X DNA loading dye

Volume of the 6X DNA loading dye: ml
(Change the volume of the solution)

Amount of Bromophenol Blue:  mg

Amount of 40% Sucrose: ml

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