Preparation of 6X DNA Loading Dye (Xylene Cyanol FF and Sucrose)

Overview

  • DNA sample is mixed with DNA loading dye prior to loading into the wells of agarose gel. 
  • DNA loading dye facilitates the loading of DNA samples into the wells of agarose gel. 
  • Usually a DNA loading dye contains at least one tracking dye (orange G, bromophenol blue or xylene cyanol FF) and a high density reagent (glycerol, sucrose or Ficoll 400). 
  • Tracking dyes help to track the progression of gel electrophoresis as well as the sample loading process into the wells of agarose gel. 
  • Two tracking dye containing loading buffers are very common for DNA gel electrophoresis. 
  • Since tracking dyes can mask the DNA fragment of interest if they co-migrate, one tracking dye containing a loading buffer can be used in such cases. In such cases, one should use a tracking dye which does not co-migrate with the DNA fragment(s) of interest. 
  • Xylene cyanol FF migrates comparatively slowly and corresponds to the migration of 4000 – 5000 bp long DNA fragments in 1% agarose gel. Due to this, Xylene cyanol FF-containing single loading dyes are not safe to monitor DNA fragments of small sizes and you may risk losing them as they can run out of the gel. 
  • 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. 
  • A 6X DNA loading dye can contain 0.03% – 0.50% (w/v) of xylene cyanol FF. High concentration of xylene cyanol FF provides very good contrast colour (light blue), which is easy to monitor upon electrophoresis progression, but it completely masks the co-migrating DNA fragments. 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
Xylene cyanol FF
Sucrose
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 
40% (w/v) sucrose 

Composition of 1X DNA loading dye 
0.042% (w/v) xylene cyanol FF 
6.67% (w/v) sucrose

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

PROCEDURE

Step 1: To prepare 10 ml of 6X DNA loading dye, weigh out 25 mg xylene cyanol FF 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:
1. 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.
2.
You can heat the solution to 60°C to dissolve the sucrose.

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. 

Note: 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 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 / Volume5 ml10 ml25 ml50 ml100 ml
Xylene cyanol FF12.5 mg25 mg62.5 mg125 mg250 mg
Sucrose2 g4 g10 g20 g40 g
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 Xylene Cyanol FF: mg
Amount of sucrose: g

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