This method uses 40% sucrose solution instead of sucrose powder. This method is convenient and easy, and does not involve adjustment of volume.
Reagents and solutions
40% (w/v) Sucrose solution (sterile)
Deionized / Milli-Q water
Equipment and disposables
15-ml screw-cap graduated polypropylene centrifuge tube
Centrifuge (for 15 ml tube)
Tube rotator (optional)
Vortex mixer (optional)
Composition of 6X DNA loading dye
0.40% (w/v) Orange G40% (w/v) sucrose
Composition of 1X DNA loading dye
0.067% (w/v) Orange G
6.67% (w/v) sucrose
Preparation of 10 ml of 6X DNA loading dye containing Orange G, and Sucrose
To prepare 10 ml of 6X DNA loading dye, weigh out 40 mg Orange G. Transfer it to a 15-mL screw-capped graduated tube. Add 10 ml of 40% sucrose solution. Mix vigorously using a vortex mixer or tube rotator to dissolve bromophenol blue and xylene cyanol FF in 40% sucrose solution.
This method is simple and convenient as it does not involve readjustment of solution volume. There will not be any significant change in the volume of the 40% sucrose solution when you dissolve small amounts of bromophenol blue in it.
We recommend using disposable nuclease nuclease-free (DNase- and RNase-free)15 ml screw-capped graduated tubes instead of beaker as all the procedure can be done in a single tube.
If you expect that Orange G can co-migrate with the DNA fragment of interest, reduce the dye concentration (add 15 -20 mg of dye). Orange G co-migrates with the 40-50 bp DNA fragment.
Store the solution in the freezer (-20°C). Solution can be stored at 2 – 8°C for a few weeks.
Sucrose-containing loading dyes are prone to mold-like growth. Avoid keeping it in the refrigerator or room temperature.
It is recommended to store the solutions in small aliquots (1 ml) at -20°C.
This solution is used for loading DNA samples onto non-denaturing agarose or polyacrylamide gels.
|Follow the table to prepare 6X agarose gel loading dye of various volumes.|
|Reagents / Volume||5 ml||10 ml||25 ml||50 ml||100 ml|
|Orange G||20 mg||40 mg||100 mg||200 mg||400 mg|
|40% Sucrose solution||5 ml||10 ml||25 ml||50 ml||100 ml|
HOW TO USE
- Take 1 part dye and 5 part DNA sample (e.g., 1μL 6X loading dye and 5μL DNA sample). Mix it and load in the well of agarose gel or polyacrylamide gel.
- If you need to take a DNA sample less than 5μL, add a water or electrophoresis buffer to bring the final volume to 6 μL.
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)