OVERVIEW
- DNA sample is mixed with DNA loading dye prior to loading onto the wells of agarose gel.
- A DNA loading dye must contain at least one tracking dyes (orange G, bromophenol blue, xylene cyanol FF or bromocresol green) and a high density reagent (glycerol, sucrose or Ficoll 400).
- A buffering agent can also be added to maintain the pH of DNA loading dye.
- DNA loading dye supplemented with a buffer, usually Tris buffer, is called DNA loading buffer.
- Tracking dyes help to track the progression of gel electrophoresis and sample loading process in the well.
- The high density reagent (glycerol, sucrose or Ficoll 400) is added to increase the density of the sample.
- Due to high density, the sample settles at the bottom of the well. It also helps a DNA sample to be confined in the well without diffusing out.
- Two tracking dyes, orange G and xylene cyanol FF, containing DNA loading buffers are very common for DNA gel electrophoresis.
- Orange G (C16H10N2Na2O7S2; Molar mass – 452.369339 gram/moleRef) is one of the most commonly used electrophoresis tracking dyes.
- Orange G, a major component in the Papanicolaou stain, is an azo dye used primarily as a histological stain.
- Orange G is available commercially as an orange crystals or powder (Color Index Number: 16230) which is highly soluble in water (solubility in water is ~ 80 mg/ml).
- Orange G usually comes as a disodium salt. The color of aqueous solution of Orange G is pH dependent.
- Orange G solution appears yellow/orange at neutral or acidic pH and red at pH greater than 9.
- Xylene cyanol FF (C25H27N2NaO6S2 ; Molar mass – 538.61 gram/mole) is available commercially as a dark green crystalline powder.
- Both tracking dyes, Orange G 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 percentage of agarose gel influences the moving position of orange G and xylene cyanol FF in the gel. The orange G and xylene cyanol FF co-migrates with ~50 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.
- Tris (Molecular Formula : C4H11NO3; Molar mass – 121.13504 gram/mole) acts as a buffering agent and maintains the pH of solution. The buffering pH range of Tris is 7.0 to 9.2 that coincides with the physiological pH of most living organisms.
- The pH value of Tris buffer is temperature and concentration dependent. For Tris buffers, pH increases about 0.025 – 0.03 unit per degree centigrade decrease in temperature, and decreases 0.03 – 0.05 unit per ten-fold dilution.
- A 6X DNA loading buffer containing orange G, Xylene cyanol FF and Ficoll 400 appears dark green in color.
- A 6X DNA loading buffer can have a wide range of Xylene cyanol [0.03% to 0.50% (w/v)] and orange G concentration [orange G concentration – 0.25 to 0.40% (w/v)]. High concentration of dye provides very good contrast colour, which is easy to monitor upon electrophoresis progression. However, dye can mask the co-migrating DNA fragments.
- The visibility of co-migrating DNA fragments decreases with increasing dye concentration, causing interference in the analysis of co-migrating DNA bands (e.g., densitometric analysis).
- Low concentration of dye 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 migrating dye bands, which sometimes disappear after a long electrophoresis run.

REQUIREMENTS
Reagents and solutions
Orange G
Xylene cyanol FF
Ficoll 400 (Suppliers list)
1M Tris.Cl, pH 7.6 at 25°C
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
0.40% (w/v) orange G
0.25% (w/v) xylene cyanol FF
15% (w/v) Ficoll 400
10 mM Tris.Cl
Composition of 1X DNA loading dye
0.067% (w/v) Orange G
0.042% (w/v) xylene cyanol FF
2.5% (w/v) Ficoll 400
1.67 mM Tris.Cl, pH 7.6
OBJECTIVE
Preparation of 10 ml of 6X DNA loading dye containing orange G, xylene cyanol FF, Ficoll 400, Tris.Cl, pH 7.6
PROCEDURE
Step 1: To prepare 10 ml of 6X DNA loading dye, weigh out 40 mg orange G, 25 mg xylene cyanol FF and 1.5 g Ficoll 400. Transfer it to a 15-mL screw-capped graduated tube. Add 7 ml deionized / Milli-Q water and 0.1 ml of 1M Tris.Cl, pH 7.6. Dissolve all ingredients by inverting the tube number of times or using a rotator/vortex mixer. You can heat the solution to 60°C (in water bath) to dissolve ingredients.
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.
Precautions:
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.
Note: The solution will appear dark green 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 or -20°C.
Tip: It is recommended to store the solution in small aliquots (1 ml).
Applications
This solution is used for loading DNA samples onto non-denaturing gels.
Follow the table to prepare 6X agarose gel loading dye of various volumes (5 ml, 10 ml, 25 ml, 100). | |||||
Reagents / Volume | 5 ml | 10 ml | 25 ml | 50 ml | 100 ml |
Orange G | 20 mg | 40 mg | 100 mg | 200 mg | 400 mg |
Xylene cyanol FF | 12.5 mg | 25 mg | 62.5 mg | 125 mg | 250 mg |
Ficoll 400 | 0.75 g | 1.5 g | 3.75 g | 7.5 g | 15 g |
1 M Tris.Cl, pH 7.6 | 0.05 ml | 0.1 ml | 0.25 ml | 0.5 ml | 1 ml |
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 |
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 Orange G: mg
Amount of Xylene Cyanol FF: mg
Amount of Ficoll 400: g
Amount of 1 M Tris (pH 7.6): ml