Preparation of 6X DNA loading dye (Bromophenol blue, xylene cyanol FF, Sucrose)

Overview

  • A DNA sample is mixed with DNA loading dye prior to loading into 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.
  • Tracking dye helps to track the progression of gel electrophoresis and sample loading process in the well.
  • 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.
  • Sucrose (C12H22O11) is a disaccharide. It is added to provide high density to the solution. 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.
  • A 6X DNA loading dye can have dye concentration ranging from 0.03% to 0.50% (W/V). A high concentration of dye provides a very good contrast color, which is easy to monitor upon electrophoresis progression. However, high dye concentration masks the co-migrating DNA fragments, causing the wrong analysis of co-migrating DNA bands (e.g., densitometric analysis). A low concentration of dye is preferred when a DNA sample is expected to contain co-migrating DNA fragment(s). However, a low concentration of dye causes a compromise in the visibility of the migrating dye band, which sometimes disappears after a long electrophoresis run.

Requirements

Reagents
> Bromophenol blue

> Xylene cyanol FF
> Sucrose
> Deionized / Milli-Q water

Equipment and disposables
> Measuring cylinder
> Conical flask / Beaker / 15 ml screw-capped tube
> Rotator

Composition of 6X DNA loading dye
> 0.25% (w/v) bromophenol blue
> 0.25% (w/v) xylene cyanol FF
> 40% (w/v) sucrose

Composition of 1X DNA loading dye
> 0.042% (W/V) bromophenol blue
> 0.042% (W/V) xylene cyanol FF
> 6.67% (w/v) sucrose

Objective
Preparation of 10 ml of 6X DNA loading dye containing bromophenol blue, xylene cyanol FF and Sucrose

Preparation:

Step 1: To prepare 10 ml of 6X DNA loading dye, weigh out 25 mg bromophenol blue, 25 mg xylene cyanol FF, and 4 g Ficoll 400. Transfer them to a screw-capped tube (graduated polypropylene centrifuge tube). Add 7 ml deionized / Milli-Q water. Mix until all ingredients dissolve completely.
Tips: We recommend using a disposable DNases-free 15-ml screw-cap graduated polypropylene centrifuge tube. Transfer all the contents and mix by inverting the tube a number of times or use a rotator. Since these tubes have milliliter marks, you can adjust solution volume without transferring the solution to the measuring cylinder. Transferring solution may not be convenient as the solution is viscous and contains dye.
Caution
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 the solvent.

Step 2: Adjust the solution volume to 10 ml with deionized / Milli-Q water. Mix it again.
Tips
1. We recommend you to give 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 -20°C for a long time. The solution can be stored at 2 – 8°C for a few weeks.

Tip: It is recommended to store the solutions in small aliquots (1 ml).

To prepare a 6X agarose gel loading dye of various volumes (5 ml, 10 ml, 25 ml, 100), follow the table.
Reagents / Volume5 ml10 ml25 ml50 ml100 ml
Bromophenol blue12.5 mg25 mg62.5 mg125 mg250 mg
Xylene cyanol FF12.5 mg25 mg62.5 mg125 mg250 mg
Sucrose2 gm4 gm10 gm20 gm40 gm
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

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