Preparation of 6X DNA Loading Lye (Bromophenol Blue and Ficoll 400)

Overview

  • DNA loading dye containing only one tracking dye bromophenol blue can be used to analyze larger DNA fragments by agarose gel electrophoresis, particularly in case where the DNA sample is expected to contain a DNA fragment(s) which can comigrate with xylene cyanol. 
  • Since comigrating DNA fragments can be masked by xylene cyanol resulting in misinterpretation of the result, avoiding xylene cyanol can be a good idea to solve this problem. In such cases, one can use only bromophenol blue containing DNA loading dye. Bromophenol blue containing gel loading dye is preferred when analyzing the large DNA fragments e.g., linearized plasmid. 
  • 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 blue 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 (agarose concentration and electrophoresis buffer) affects 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 can 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 bromophenol blue 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 the migrating dye band, which sometimes disappears after a long electrophoresis run. 
  • Ficoll 400 is a high molecular weight, neutral, hydrophilic, polysaccharide. It is added to provide high density to the sample. 
  • 6X DNA loading dye containing bromophenol blue and Ficoll 400 is blue in color. 
  • DNA sample appears blue in colour when it is mixed with bromophenol blue containing loading dye. Due to the blue colour of the sample, the loading process in the wells of agarose gel can be monitored easily.

REQUIREMENTS

Reagents and solutions
Bromophenol blue
Ficoll 400
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
15% (w/v) Ficoll 400

Composition of 1X DNA loading dye 
0.042% (w/v) bromophenol blue
1.5% (w/v) Ficoll 400

Objective 
Preparation of 10 ml of 6X DNA loading dye containing bromophenol blue and Ficoll 400

PROCEDURE

Step 1: To prepare 10 ml of 6X DNA loading dye, weigh out 25 mg bromophenol blue and 1.5 g Ficoll 400. 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:
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.

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.

Step 2: Adjust the volume to 10 ml with deionized / Milli-Q water. Mix it again. 
Bring all the solution down by centrifuging the tube briefly and then adjust the volume 10 ml.

Note: The solution will appear dark blue/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 at room temperature or 4°C.

Note:
Ficoll-containing loading dyes are stable at room temperature.
 
Tip:
It is recommended to store the solutions in small aliquots (1 ml).

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

Follow the table to prepare 6X agarose gel loading dye of various volumes (5 ml, 10 ml, 25 ml, 100).
Reagents / Volume5 ml10 ml25 ml50 ml100 ml
Bromophenol blue12.5 mg25 mg62.5 mg125 mg250 mg
Ficoll 400
0.75 g1.5 g3.75 g7.5 g15 g
WaterAdjust the final volume to 5 mlAdjust the final volume to 10 mlAdjust the final volume to 25 mlAdjust the final volume to 50 mlAdjust the final volume to 100 ml

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 Ficoll 400: g

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