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

Overview

  • DNA sample is mixed with DNA loading dye prior to loading into the wells of agarose gel.
  • A DNA loading dye must contain at least one dye (orange G, bromophenol blue, xylene cyanol FF or bromocresol green) and a high density reagent (glycerol, sucrose or Ficoll 400).
  • Dyes help to track the progression of gel electrophoresis and sample loading process in the well.
  • Two dyes containing DNA loading dye is very common for DNA gel electrophoresis. The most common dyes are bromophenol blue and xylene cyanol FF.
  • Bromophenol blue migrates fast in the agarose gel and corresponds to the migration of 300 – 500 bp long DNA fragment in 1% agarose gel.
  • Xylene cyanol FF migrates comparatively slow and corresponds to the migration of 4000 – 5000 bp long DNA fragment in 1% agarose gel.
  • A 6X DNA loading dye can have dye concentration ranging from 0.03% to 0.50% (W/V). High concentration of dye provides very good contrast colour, which is easy to monitor upon electrophoresis progression. However, high dye concentration masks the co-migrating DNA fragments, causing wrong analysis of co-migrating DNA bands (e.g., densitometric analysis). Low concentration of dye is preferred when 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 band, which sometime disappear after a long electrophoresis run.
  • Sucrose is added to provide high density to solution. Due to high density, sample settle at the bottom of the well. It also helps DNA sample to be confined in the well without diffusing out.

Requirement

Reagents:

  • Bromophenol blue
  • Xylene cyanol FF
  • Sucrose
  • Deionized / Milli-Q water

Equipments and disposables:

  • Measuring cylinder
  • Conical flask / Beaker / 15 ml screw-capped tube
  • Rotator

Composition

  • 0.25% (W/V) bromophenol blue
  • 0.25% (W/V) xylene cyanol FF
  • 40% (W/V) sucrose

Objective

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


Preparation:

  • To prepare 10 ml of 6X DNA loading dye, weigh out 25 mg bromophenol blue, 25 mg xylene cyanol FF and 4 gm Sucrose. Transfer them to 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 nuclease-free (DNase- and RNase-free) 15 ml screw-capped tube with millilitre marks. Transfer all the contents in it. Add 7 ml deionized / Milli-Q water and mix the content by inverting the tube number of times or using rotator. Once the content is mixed, adjust the volume to 10 ml. 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 is viscous and contains dye.

Precautions:

  1. Do not dissolve in 10 ml of deionized / Milli-Q water. In most cases, solution volume increases when the large amount of solute dissolves in solvent.
  2. Use nuclease-free, autoclaved deionized / Milli-Q water and glasswares.
  • Adjust the volume to 10 ml with deionized / Milli-Q water. Mix it again.

     

Storage

  • Store the solution at -20°C for long time. Solution can be stored at 2 – 8°C for few weeks.

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


 

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 cynol FF 12.5 mg 25 mg 62.5 mg 125 mg 250 mg
Sucrose 2 gm 4 gm 10 gm 20 gm 40 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