Preparation of 6X DNA Loading Dye (Orange G and Sucrose)

Overview

• DNA loading dye containing only one tracking dye, Orange G, can be used to analyze large and moderate size DNA fragments by agarose gel electrophoresis, particularly when the DNA sample is expected to contain DNA fragment(s) that can be masked by xylene cyanol and bromophenol blue. 
• In such situations, Orange G-containing loading dye can be a good choice to avoid such problems.
• Orange G (C16H10N2Na2O7S2Ref; Molar mass – 452.369339 gram/mole) 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). It 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. 
• The composition of agarose gel defines the moving position of Orange G in the gel. Orange G co migrates with ~40 bp (1 x TBE) or ~60 bp (1 x TAE) fragments in 1% agarose gel. 
• A 6X DNA loading dye may contain 0.15% – 0.50% (w/v) of Orange G. 
• Sucrose is added to provide high density to solution. Due to high density, the sample settles at the bottom of the well. It also helps DNA samples to be confined in the well without diffusing out from the well.

REQUIREMENTS

Reagents and solutions
Sucrose
Ficoll 400
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
40% (w/v) Sucrose

Composition of 1X DNA loading dye 
0.067% (w/v) Orange G
6.67% (w/v) Ficoll 400

Objective 
Preparation of 10 ml of 6X DNA loading dye containing Orange G and Sucrose

PROCEDURE

Step 1: To prepare 10 ml of 6X DNA loading dye, weigh out 40 mg Orange G and 4 g Sucrose. Transfer it to a 15-mL screw-capped graduated tube. Add 7 ml deionized / Milli-Q water. Dissolve the content by inverting the tube number of times or using a rotator/vortexer until all the ingredients are dissolved completely.

Tips:
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.

Precaution:
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. 
Bring all the solution down by centrifuging the tube briefly and then adjust the volume 10 ml.

Note: The solution will appear pink-orange 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.

Storage:
Store the solution in the freezer (-20°C).

Precaution:
You can not keep the sucrose-containing loading dyes at 4°C for a long time as they are prone to fungal growth.

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

Applications
This solution is used for loading DNA samples into 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)

Click

Amount of Orange G: mg
Amount of Sucrose: g