Preparation of 6X DNA Gel loading buffer (Bromophenol blue, Orange G, Xylene Cyanol FF, EDTA and Ficoll 400)

OVERVIEW

A 6x DNA Gel loading buffer containing Orange G, Bromophenol blue, Xylene cyanol FF, EDTA and Ficoll 400 is prepared by dissolving 25 mg bromophenol blue, 25 mg xylene cyanol FF, 40 mg Orange G, 1.5 g Ficoll 400 and 1.2 ml of 0.5 M EDTA in 10 mM Tris.Cl, pH 7.6 to a final volume of 10 ml. The final solution (6x DNA loading dye) will contain 0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol FF, 15% (w/v) Ficoll 400, 60 mM EDTA and 10 mM Tris.

PROS AND CONS

Pros

  • Stable at room temperature, can be stored at room temperature
  • Contrast colour of tracking dyes especially bromophenol blue and xylene cyanol FF helps to monitor sample loading process and progression of electrophoresis.
  • Three-colour loading dyes are a better indicator of resolution of DNA fragments. By looking at separation among tracking dyes on agarose gel, one can guess if the sufficient separation of DNA fragments of interest has been achieved. 
  • Ficoll 400-containing DNA loading dye sinks DNA samples in agarose well better than glycerol and sucrose-containing loading dye.
  • EDTA stops nuclease action by sequestering divalent metal ions
  • Tris-containing loading buffers resist pH fluctuations

Cons

  • The cost of Ficoll 400 is much more than glycerol and sucrose
  • Each tracking dye costs money
  • Bromophenol blue and xylene cyanol can mask the comigrating DNA fragments

Applications

  • DNA samples loading onto non-denaturing agarose or polyacrylamide gels
  • Suitable for the analysis of PCR reaction products by agarose gel electrophoresis (primer dimers run close to Orange G position)
  • A better alternative for restriction enzyme digested DNA 

REQUIREMENTS

Reagents and solutions
Bromophenol blue
Xylene cyanol FF
Orange G
0.5 M EDTA (sterile, autoclaved)
1 M Tris.Cl, pH 7.6 at 25°C (sterile, autoclaved)
Ficoll 400
Deionized / Milli-Q water (sterile, autoclaved)

Equipment and disposables
15-ml screw-cap graduated polypropylene centrifuge tube *
Tube Rotator (optional)

* We recommend using a disposable Nuclease-free 15-ml screw-cap graduated polypropylene centrifuge tube. These tubes are very convenient to prepare 10 ml solution as the whole process can be done in a single tube without transferring solution to another vial. Transferring solutions may not be convenient as the solution will be viscous and will have dye. If you do not have a 15-ml graduated polypropylene centrifuge tube, you can use a beaker or flask.

COMPOSITION

Composition of 6X DNA loading dye
0.40% (w/v) orange G
0.25% (w/v) bromophenol blue
0.25% (w/v) xylene cyanol FF
15% (w/v) Ficoll 400
60 mM EDTA
10 mM Tris.Cl, pH 7.6

Composition of 1X DNA loading dye
0.067% (w/v) orange G
0.042% (w/v) bromophenol blue
0.042% (w/v) xylene cyanol FF
2.5% (w/v) Ficoll 400
10 mM EDTA
1.67 mM Tris.Cl, pH 7.6

OBJECTIVE

Preparation of 10 ml of 6x DNA loading dye containing orange G, bromophenol blue, xylene cyanol FF, Ficoll 400 and EDTA

PREPARATION

Step 1: To prepare 10 ml of 6x DNA loading dye, weigh out 1.5 g Ficoll 400 and transfer it to a screw-capped tube (graduated polypropylene centrifuge tube). Add 6 ml deionized / Milli-Q water, 1.2 ml of 0.5 M EDTA, and 0.1 ml of 1M Tris.Cl, Ph 7.6. Dissolve Ficoll 400 by inverting the tube a number of times or using a tube rotator. You can gently heat the solution by keeping the vial to 60°C water bath.

Note
The solution will appear clear colorless to yellowish hazy.

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

Step 2: Add 40 mg orange G, 25 mg bromophenol blue and 25 mg xylene cyanol FF, in Ficoll 400 solution (prepared in step 1). Dissolve them by inverting the tube a number of times or using a tube rotator. 

Note
Depending on your requirements, you can change the concentration of tracking dyes (0.03% to 0.50% (w/v) for bromophenol blue and xylene cyanol FF and 0.15% – 0.50% (w/v) for Orange G). The high concentration provides a good contrast color, which is easy to monitor upon electrophoresis progression but also interfere in the analysis of comigrating DNA bands (e.g., densitometric analysis) by masking them. A low concentration of tracking dye is preferred when a DNA sample is expected to contain co-migrating DNA fragment(s). The low concentration of tracking dye causes a compromise in the visibility of migrating tracking dye bands, which sometimes disappears after a long electrophoresis run.

Step 3: Adjust the solution volume to 10 ml with deionized / Milli-Q water. Mix it again.

Tips

  1. We recommend giving a short spin to a 15 ml tube just before adjusting the final volume to 10 ml. Short spin will help to collect all solutions which are adhered to sides and the lid of the tube.
  2. The solution will appear dark purple 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 in small aliquots of 1 ml in 1.5 ml eppendorf microcentrifuge tubes. You can store the solution at 4°C. Working aliquot can be stored at room temperature, the rest of them store at 4°C or freeze them. 

Follow the table to prepare 6x DNAloading dye of various volumes.
Reagents / Volume5 ml10 ml25 ml50 ml100 ml
Bromophenol blue12.5 mg25 mg62.5 mg125 mg250 mg
Xylene cyanol FF12.5 mg25 mg62.5 mg125 mg250 mg
Orange G20 mg40 mg100 mg200 mg400 mg
Ficoll 4000.75 g1.5 g3.75 g7.5 g15 g
0.5M EDTA
0.6 ml1.2 ml
3 ml
6 ml
12 ml
1M Tris.Cl pH 7.6
0.05 ml
0.1 ml
0.25 ml
0.5 ml
1 ml
WaterAdjust the final volume to 5 mlAdjust the final volume to 10 mlAdjust the final volume to 25 mlAdjust the final volume to 50 mlAdjust 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 Bromophenol Blue: 25 mg
Amount of Xylene Cyanol FF: 25 mg
Amount of Orange G: 40 mg
Amount of Ficoll: 1.5 g
Amount of 0.5M EDTA: 1.2 ml
Amount of 1M Tris.Cl pH 7.6: 0.1 ml

 

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