OVERVIEW

A 6x DNA loading dye containing Orange G, Bromophenol blue, Xylene cyanol FF, and Ficoll 400 is prepared by dissolving 25 mg bromophenol blue, 25 mg xylene cyanol FF, and 40 mg Orange G in 10 ml of 15% aqueous solution of Ficoll 400. The final solution (6x DNA loading dye) will contain 0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol FF, and 15% (w/v) Ficoll 400.

PROS AND CONS

Pros

• Stable at room temperature, can be stored at room temperature
  
• Contrast colour of tracking dyes especially bromophenol blue and xylene cyanol FF helps to monitor sample loading process and progression of electrophoresis.
• Three-colour loading dyes are a better indicator of resolution of DNA fragments. By looking at separation among tracking dyes on agarose gel, one can guess if the sufficient separation of DNA fragments of interest has been achieved. 
• Ficoll 400-containing DNA loading dye sinks DNA samples in agarose well better than glycerol and sucrose-containing loading dye.

Cons

• The cost of Ficoll 400 is much more than glycerol and sucrose
• Each tracking dye costs money
• Bromophenol blue and xylene cyanol can mask the comigrating DNA fragments

Applications

• DNA samples loading onto non-denaturing agarose or polyacrylamide gels
• Suitable for the analysis of PCR reaction products by agarose gel electrophoresis (primer dimers run close to Orange G position)

REQUIREMENTS

Reagents and solutions
Bromophenol blue
Xylene cyanol FF
Orange G
15% Ficoll 400 solution in water (autoclaved)

Equipment and disposables
15-ml screw-cap graduated polypropylene centrifuge tube *
Tube Rotator (optional)

* We recommend using a disposable Nuclease-free 15-ml screw-cap graduated polypropylene centrifuge tube. These tubes are very convenient to prepare 10 ml solution as the whole process can be done in a single tube without transferring solution to another vial. Transferring solutions may not be convenient as the solution will be viscous and will have dye. If you do not have a 15-ml graduated polypropylene centrifuge tube, you can use a beaker or flask.

COMPOSITION

Composition of 6X DNA loading dye
0.40% (w/v) orange G
0.25% (w/v) bromophenol blue
0.25% (w/v) xylene cyanol FF
15% (w/v) Ficoll 400

Composition of 1X DNA loading dye
0.067% (w/v) orange G
0.042% (w/v) bromophenol blue
0.042% (w/v) xylene cyanol FF
2.5% (w/v) Ficoll 400

OBJECTIVE

Preparation of 10 ml of 6x DNA loading dye containing orange G, bromophenol blue, xylene cyanol FF and Ficoll 400

PREPARATION

To prepare 10 ml of 6x DNA loading dye, weigh out 40 mg orange G, 25 mg bromophenol blue and 25 mg xylene cyanol FF and transfer it to a screw-capped tube (graduated polypropylene centrifuge tube). Add 10 ml of 15% Aqueous solution of Ficoll 400. Dissolve all ingredients by inverting the tube a number of times or using a tube rotator.

Note
Depending on your requirements, you can change the concentration of tracking dyes (0.03% to 0.50% (w/v) for bromophenol blue and xylene cyanol FF and 0.15% – 0.50% (w/v) for Orange G). The high concentration provides a good contrast color, which is easy to monitor upon electrophoresis progression but also interfere in the analysis of comigrating DNA bands (e.g., densitometric analysis) by masking them. A low concentration of tracking dye is preferred when a DNA sample is expected to contain co-migrating DNA fragment(s). The low concentration of tracking dye causes a compromise in the visibility of migrating tracking dye bands, which sometimes disappears after a long electrophoresis run.

Tip
The solution will appear dark purple in color with no undissolved particles. If there are any undissolved particles in the solution, remove them by centrifuging the tube at 4000 – 5000 rpm for 10 min at room temperature.

STORAGE

Store the solution in small aliquots of 1 ml in 1.5 ml eppendorf microcentrifuge tubes. You can store the solution at 4°C or -20 °C. Working aliquot can be stored at room temperature.

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)

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Amount of Bromophenol Blue: 25 mg
Amount of Xylene Cyanol FF: 25 mg
Amount of Orange G: 40 mg
Amount of 15% Ficoll solution: 10 ml