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

 Overview

  • 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.
  • 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.
  • 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 move faster than xylene cyanol in agarose gel in spite of its higher molecular weight.
  • The composition of agarose gel affects 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 sample.
  • 6X DNA loading dye containing bromophenol blue, Xylene cyanol FF and Ficoll 400 appears dark blue/ purple in color.
  • A 6X DNA loading dye can have tracking dye concentration ranging from 0.03% to 0.50% (W/V). High concentration provides very good contrast colour, which is easy to monitor upon electrophoresis progression. However, high tracking dye concentration masks the co-migrating DNA fragments, and interfere in the analysis of co-migrating DNA bands (e.g., densitometric analysis). Low concentration of tracking dye is preferred when DNA sample is expected to contain co-migrating DNA fragment(s). However, low concentration of tracking dye causes a compromise in the visibility of migrating dye band, which sometime disappear after a long electrophoresis run.

Requirement

  • Reagents
    • Bromophenol blue
    • Xylene cyanol FF
    • Ficoll 400
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker / 15 ml screw-capped tube
    • Magnetic stirrer (optional)

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 gram Ficoll 400. Transfer them to a screw-capped tube (15 ml with millilitre marks). Add 7 ml deionized / Milli-Q water. Mix until all ingredients are dissolved completely.

Tips:

  • We recommend to use disposable DNases-free 15 ml screw-capped tube with millilitre marks. Transfer all the contents and mix by inverting the tube number of times or use rotator. Since the tube is marked, you don’t need to transfer the content to measuring cylinder. Transferring solution may not be convenient as the solution contains dye.

Precautions:

  • 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.

Tips:

  • We recommend you to give a short spin to 15 ml tube just before adjusting the volume 10 ml. Short spin will help to collect all solution which are adhered to sides and lid of the tube.
  • The solution will appear dark blue/purple in colour with no undissolved particles. If there is 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 long time.

Tips:

  • Aliquot 1 ml in 1.5 ml eppendorf microcentrifuge tubes.

Applications

  • This solution is used for loading DNA samples onto nondenaturing gels.
To prepare 6X agarose gel loading dye of various volume (5 ml, 10 ml, 25 ml, 100), follow the table.
Reagents / Volume 5 ml 10 ml 25 ml 50 ml 100 ml
Bromophenol blue 12.5 mg 25 mg 62.5 mg 125 mg 250 mg
Xylene cyanol FF 12.5 mg 25 mg 62.5 mg 125 mg 250 mg
Ficoll 400 0.75 gm 1.5 gm 3.75 gm 7.5 gm 15 gm
Water Adjust the final volume to 5 ml Adjust the final volume to 10 ml Adjust the final volume to 25 ml Adjust the final volume to 50 ml Adjust the final volume to 100 ml