Preparation of 6X DNA Loading Dye (Bromophenol blue, Xylene Cyanol FF, Ficoll 400)

Overview

• A DNA sample is mixed with DNA loading dye prior to loading onto the wells of agarose gel.
• Two tracking dyes-containing DNA loading dye is very common for DNA gel electrophoresis. The most common tracking dyes are bromophenol blue and xylene cyanol FF.
• 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 the 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.
• Xylene cyanol FF (C₂₅H₂₇N₂NaO₆S₂ ; Molar mass – 538.61 gram/mole) is available commercially as a dark green crystalline powder.
• Both tracking dyes, Bromophenol blue and Xylene cyanol FF, are soluble in water (solubility in water is ~ 1 mg/ml) and carry net negative charge at neutral or slightly basic pH (the pH of the electrophoresis buffer). The net negative charge on Xylene cyanol FF is less than Bromophenol blue. Because of this, Bromophenol blue moves faster than xylene cyanol in agarose gel in spite of its higher molecular weight.
• The composition of agarose gel affects the moving position of bromophenol blue and xylene cyanol FF in the gel. The bromophenol blue and xylene cyanol FF co-migrates with ~300 bp and ~4000 bp DNA fragments in 1% agarose gel respectively.
• Ficoll 400 is a high molecular weight, neutral, hydrophilic, polysaccharide. It is added to provide high density to the sample. Due to high density, samples settle at the bottom of the well. It also helps DNA samples to be confined in the well without diffusing out.
• 6X DNA loading dye containing bromophenol blue, Xylene cyanol FF and Ficoll 400 appears dark blue/ purple in color.

REQUIREMENTS

Reagents and solutions
Bromophenol blue
Xylene cyanol FF
Ficoll 400
Deionized / Milli-Q water

Equipment and disposables
15-ml screw-cap graduated polypropylene centrifuge tube
Tube Rotator

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

Composition of 1X DNA loading dye
0.042% (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 bromophenol blue, xylene cyanol FF and Ficoll 400

PREPARATION

Step 1: To prepare 10 ml of 6X DNA loading dye, weigh out 25 mg bromophenol blue, 25 mg xylene cyanol FF, and 1.5 g Ficoll 400. Transfer it to a 15-mL screw-capped graduated centrifuge tube. Add 7 ml deionized / Milli-Q water. Dissolve the content by inverting the tube number of times or using a rotator/vortex mixer until all the ingredients are dissolved completely.

Note:
The concentrations of bromophenol blue and xylene cyanol FF in the 6x DNA loading dye can vary from 0.03% to 0.50% (w/v). The high concentration provides a very good contrast color, which is easy to monitor upon electrophoresis progression. However, high tracking dye concentration masks and interferes in the analysis of co-migrating DNA fragments (e.g., densitometric analysis). A low concentration of tracking dye is preferred when a DNA sample is expected to contain co-migrating DNA fragment(s). However, the low concentration of tracking dye causes a compromise in the visibility of a migrating tracking dye band, which sometimes disappears after a long electrophoresis run.

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

Step 2: Adjust the solution volume to 10 ml with deionized / Milli-Q water. Mix it again.
Tips
1. We recommend you giving a short spin to a 15 ml tube just before adjusting the volume 10 ml. Short spin will help to collect all solutions which are adhered to sides and the lid of the tube.
2. 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 for a long time.
Tip: It is recommended to store the solutions in small aliquots (1 ml).

Applications
This solution is used for loading DNA samples onto nondenaturing 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 Xylene Cyanol FF: mg

Amount of Ficoll: g