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

Overview

• DNA sample is mixed with the DNA loading dye prior to loading into the wells of agarose gel. A DNA loading dye must contain at least one tracking dye (orange G, bromophenol blue, xylene cyanol FF or bromocresol green) and a high density reagent (glycerol, sucrose or Ficoll 400).
• Tracking dyes help to track the progression of gel electrophoresis as well as to monitor the sample loading process in the well.
• The high density reagent (glycerol, sucrose or Ficoll 400) is added to increase the density of the sample. Due to high density, the sample settles at the bottom of the well. It also helps the DNA sample to be confined in the well without diffusing out from the well.
• Bromophenol Blue (C19H10Br4O5S ; Molar mass – 669.96 gram/mole) is one of the most commonly used electrophoresis tracking dyes and is structurally related to phenolphthalein.
• Bromophenol blue is available commercially as a light pink to purple crystalline powder which is readily soluble in water (solubility in water is ~ 1 mg/ml) and other organic solvents e.g., ethanol, DMSO, and dimethyl formamide (DMF).
• 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 is a weak acid. It carries negative charge at neutral or slightly basic pH (the pH of the electrophoresis buffer). Therefore it migrates toward the positive electrode.
• The composition of agarose gel controls the moving position of bromophenol blue in the gel. The bromophenol blue co-migrates with ~ 300 bp DNA fragments in 1% agarose gel.
• A 6X DNA loading dye may contain 0.03% – 0.50% (w/v) of bromophenol blue. High concentration of bromophenol blue provides very good contrast color (light blue), which is easy to monitor upon electrophoresis progression, but it completely masks the co-migrating DNA fragments. Low concentration of dye is preferred when a DNA sample is expected to contain co-migrating DNA fragment(s). However, low concentration of dye causes a compromise in the visibility of migrating dye bands, which sometimes disappear after a long electrophoresis run.
• Bromophenol blue-containing gel loading dye is preferred when analyzing the large DNA fragments e.g., plasmids.
• 6X DNA loading dye containing bromophenol blue and sucrose appears blue in color.
• Sucrose (C₁₂H₂₂O₁₁), a disaccharide, is composed of the monosaccharides glucose and fructose. It is added to provide high density to the sample.

REQUIREMENTS

Reagents and solutions
Bromophenol blue
Sucrose
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
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

PROCEDURE

Step 1: To prepare 10 ml of 6X DNA loading dye, weigh out 25 mg Bromophenol blue and 4 g sucrose. Transfer it to a 15-mL screw-capped graduated tube. Add 7 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.

Tips:
1. We recommend using a disposable nuclease-free 15-mL screw-capped graduated tube. It allows you to complete the whole process in a single tube without transferring liquid to the measuring cylinder. If you use a beaker or conical flask, you need to transfer the content to a measuring cylinder to adjust the volume to 10 ml. Transferring solution may not be convenient as the solution will be viscous and contains dye.
2. You can heat the solution to 60°C to dissolve the sucrose.

Precaution:
1. Do not dissolve in 10 ml of deionized / Milli-Q water. In most cases, solution volume increases when a large amount of solutes dissolve in a solvent.
2. Use nuclease-free, autoclaved deionized / Milli-Q water and glasswares.
3. 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.

Step 2: Adjust the volume to 10 ml with deionized / Milli-Q water. Mix it again. 

Note: If there are any undissolved particles in the solution, remove them by centrifuging the tube at 4000 – 5000 rpm for 10 min.

Storage:
Store the solution in the freezer (-20°C).

Precaution:
You can not keep the sucrose-containing loading dyes at 4°C for a long time as they are prone to fungal growth. 
Tip:
It is recommended to store the solutions in small aliquots (1 ml).

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

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: mg
Amount of sucrose: g