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.

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 Orange G: mg
Amount of Xylene Cyanol FF: mg
Amount of Ficoll 400: g
Amount of 1 M Tris (pH 7.6): ml