Preparation of 6X DNA Loading Dye (Bromophenol Blue and Glycerol)

Overview

• DNA loading dye containing only one tracking dye Bromophenol blue (BPB) can be used to analyze larger DNA fragments by agarose gel electrophoresis, particularly in case where the DNA sample is expected to contain DNA fragment(s) which can co-migrate with xylene cyanol FF.
• Since masking of co-migrating DNA fragments by xylene cyanol FF can mislead the interpretation of experiment, avoiding xylene cyanol FF can be a good idea to solve this problem. In such cases, one can use only Bromophenol blue (BPB) containing DNA loading dye.
• Bromophenol blue (C19H10Br4O5S ; Molar mass – 669.96 gram/mole) is a weak acid. It is available commercially as a light pink to purple crystalline powder.
• The color of aqueous solution of bromophenol blue is pH dependent. Bromophenol blue solution appears yellow at pH 3.0, purple at pH 4.6, and blue at neutral pH.
• Bromophenol blue is soluble in water (solubility in water is ~ 1 mg/ml) and carries net negative charge at neutral or slightly basic pH (the pH of the electrophoresis buffer).
• The composition of agarose gel defines the moving position of bromophenol blue in the gel. The bromophenol blue co-migrates with ~300 bp DNA fragments in 1% agarose gel.
• Glycerol (C3H8O3), a sugar alcohol, is a simple polyol compound. It is added to provide high density to the sample.
• 6X DNA loading dye containing bromophenol blue and glycerol appears purple in color.
• A 6X DNA loading dye can have bromophenol blue concentration ranging from 0.03% to 0.50% (w/v). High concentration of bromophenol blue provides very good contrast colour, which is easy to monitor upon electrophoresis progression. However, high concentration masks the co-migrating DNA fragments, and interfere in the analysis of co-migrating DNA bands (e.g., densitometric analysis). Low concentration of bromophenol blue is preferred when a DNA sample is expected to contain co-migrating DNA fragment(s). However, low concentration of tracking dye causes a compromise in the visibility of the migrating dye band, which sometimes disappears after a long electrophoresis run.

REQUIREMENTS

Reagents and solutions
Bromophenol blue
85% Glycerol
Deionized / Milli-Q water

Equipments 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
60% (V/v) Glycerol

Composition of 1X DNA loading dye
0.042% (w/v) bromophenol blue
10% (w/v) Glycerol

Objective
Preparation of 10 ml of 6X DNA loading dye containing bromophenol blue and Glycerol

PROCEDURE

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 7.06 ml of 85% Glycerol and 2.94 ml deionized / Milli-Q water. Dissolve the content by inverting the tube number of times or using a rotator/vortexer until all the ingredients are dissolved completely.

Note:
Commercially available glycerol can have variable percentages ranging from 85% to 100%. Any of them can be used for preparation of DNA loading dye. You need to calculate the amount of glycerol and water if you are using different percentages of glycerol.

Precaution:
Use nuclease-free, autoclaved deionized / Milli-Q water and glasswares.

Storage:
Store the solution at -20°C for a long time. Solution can be stored at room temperature for a few weeks.

Tip:
It is recommended to store the solutions in small aliquots (1 ml).
Spin the tube at 5000 rpm for 1 min to bring all content to the bottom of the tube. Aliquot 1 ml in 1.5 ml eppendorf microcentrifuge tubes.

Applications
This solution is used for loading DNA samples into non-denaturing gels.