Preparation of 6X DNA Loading Lye (Xylene Cyanol FF and Glycerol)

Overview

• DNA loading dye containing only one tracking dye, xylene cyanol FF, can be used to analyze smaller DNA fragments by agarose gel electrophoresis, particularly in case where the DNA sample is expected to contain a DNA fragment(s) which can comigrate with bromophenol blue.
• Since masking of comigrating DNA fragments by bromophenol blue can cause wrong interpretation of experiment, avoiding bromophenol blue can be a good idea to solve this problem. In such cases, either one can use only Xylene cyanol FF containing DNA loading dye or DNA loading dye containing xylene cyanol FF and Orange G.
• Xylene cyanol FF (C25H27N2NaO6S2 ; Molar mass – 538.61 gram/mole) is available commercially as a dark green crystalline powder.
• Xylene cyanol FF is soluble in water (solubility in water is ~ 1 mg/ml) and carries net negative charge at neutral or slightly basic pH (the pH of the electrophoresis buffer).
• The composition of agarose gel defines the moving position of xylene cyanol FF in the gel. The xylene cyanol FF co-migrates with ~4000 bp DNA fragments in 1% agarose gel.
• Since xylene cyanol FF comigrates with ~4000 bp DNA fragments, the position of smaller DNA fragments in agarose gel can be difficult to predict during the gel run. This can cause the smaller DNA fragments to run out from the gel. To avoid this problem, one should monitor the agarose gel in the gel documentation system/uv transilluminator once the xylene cyanol FF reaches 1/3 of the agarose gel.
• A 6X DNA loading dye can have xylene cyanol FF concentration ranging from 0.03% to 0.50% (W/V). High concentration of xylene cyanol FF provides very good contrast colour, which is easy to monitor upon electrophoresis progression. However, high 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 a DNA sample is expected to contain co-migrating DNA fragment(s). However, low concentration of xylene cyanol FF causes a compromise in the visibility of migrating the dye band, which sometimes disappears after a long electrophoresis run.
• 6X DNA loading dye containing Xylene cyanol FF and glycerol appears dark blue in color. DNA sample appears blue in colour when it is mixed with Xylene cyanol FF containing loading dye. Due to the blue colour of the sample, the loading process in the wells of agarose gel can be monitored easily.
• Glycerol (C3H8O3), a sugar alcohol, is a simple polyol compound. It is added to provide high density to the sample. Due to high density, the sample settles at the bottom of the well without diffusing out.

REQUIREMENTS

Reagents and solutions
Xylene cyanol FF
85% Glycerol
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
0.25% (w/v) xylene cyanol FF 
60% (V/v) Glycerol

Composition of 1X DNA loading dye 
0.042% (w/v) xylene cyanol FF 
10% (w/v) Glycerol

Objective 
Preparation of 10 ml of 6X DNA loading dye containing xylene cyanol FF and Glycerol

PROCEDURE

To prepare 10 ml of 6X DNA loading dye, weigh out 25 mg xylene cyanol FF. Transfer it to a 15-mL screw-capped graduated tube. Add 7.06 ml of 85% Glycerol and 2.94 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.

Note:
Commercially available glycerol can have variable percentages ranging from 85% to 100%. Any of them can be used for preparation of DNA loading dye. You need to calculate the amount of glycerol and water if you are using different percentages of glycerol.

Precaution:
Use nuclease-free, autoclaved deionized / Milli-Q water and glasswares.

Storage:
Store the solution at -20°C for a long time. Solution can be stored at room temperature for a few weeks. 

Tip:
It is recommended to store the solutions in small aliquots (1 ml).
Spin the tube at 5000 rpm for 1 min to bring all content to the bottom of the tube. Aliquot 1 ml in 1.5 ml eppendorf microcentrifuge tubes.

Applications
This solution is used for loading DNA samples into non-denaturing gels.