Jun 24, 2022

Public workspaceProduction of 6x DNA Loading Dye

This protocol is a draft, published without a DOI.
  • 1Beneficial Bio, Mboalab
Nadine Mowoh
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Protocol Citation: Stephane Fadanka, Nadine Mowoh 2022. Production of 6x DNA Loading Dye. protocols.io https://protocols.io/view/production-of-6x-dna-loading-dye-cbdcsi2w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: June 15, 2022
Last Modified: June 24, 2022
Protocol Integer ID: 64644
Keywords: preparing 6x DNA loading dye, preparing DNA tracking dye
Abstract
The process of gel electrophoresis involves separating DNA fragments using an electrical current while tracking the rate of molecular movement through a filtering gel.
Adding blue or orange tracking dye to colourless DNA samples allows you to see your sample and obtain information about how DNA molecules move during electrophoresis. Identification is based on the size of DNA bands on the gel after migration of molecules.

The Beneficial Bio 6x loading dye is composed of bromophenol blue and xylene cyanol. Thus, it helps you determine the rate of movement of different size DNA molecules in the gel during electrophoresis.
When composing or preparing DNA loading dyes, a heavy, syrupy substance that gives more density to the DNA sample before it is inserted in the wells is needed. This will help the DNA sample sink forming a layer in the well instead of being dispersed in the absence of a heavy substance. In this formulation, trehalose is used to give the DNA samples the desired viscosity.

Materials
ReagentTrehalose in a final concentration of 14.48%
ReagentSDS in a final concentration of 0.3%
ReagentNa2EDTASigma – Aldrich
ReagentGlycerolSigma – Aldrich in a final concentration of 23.4%
ReagentXylene cyanolBio Basic Inc.
ReagentBromophenol blueBio Basic Inc.
Electronic or sensitive weighing balance
Measuring cylinder
15ml Eppendorf tube

Protocol materials
ReagentTrehalose
Materials, Step 1
ReagentSDS
Materials, Step 2
ReagentNa2EDTAMerck MilliporeSigma (Sigma-Aldrich)
Materials, Step 3
ReagentGlycerolMerck MilliporeSigma (Sigma-Aldrich)
Materials
ReagentGlycerolMerck MilliporeSigma (Sigma-Aldrich)
Step 4
ReagentXylene cyanolBio Basic Inc.
Materials, Step 4
ReagentBromophenol blueBio Basic Inc.
Materials, Step 4
Safety warnings
All reagents and chemicals used here are generally safe but it is advisable to always wear protective clothing to avoid accidental spills or splashes.
Before start
Check to make sure you have all the starting materials available and assembled to prepare the reagent stocks.
Preparation of Reagent stocks
Preparation of Reagent stocks
10m
10m

Note
In order to produce a 6x DNA loading dye, we begin by preparing reagent stocks and weighing dye powders to constitute a concentrated dye stock (could be called a 60x stock) which is later diluted to make the 6x Dye stock.


Preparing Concentration20 % (v/v) Trehalose (100ml)

  • Accurately weigh Amount20 g of ReagentTrehaloseSigma Aldrich powder into a Amount500 mL beaker
  • Use a measuring cylinder to measureAmount100 mL of sterile distilled water into the beaker and stir gently until the powder is completely dissolved.
  • Transfer the content into a clean Duran bottle, cork and keep at Temperature4 °C until used.


Preparing Concentration10 % (v/v) ReagentSDSSigma Aldrich (100ml)

  • Accurately weigh Amount10 g of SDS powder into a Amount500 mL beaker
  • Use a measuring cylinder to measure Amount100 mL of sterile distilled water into the beaker and stir gently until the powder is completely dissolved.
  • You may heat it up a little up to 40c to 60c to ease dissolution but do not autoclave as it may result in precipitation.
  • Transfer the content into a clean Duran bottle, cork and store at TemperatureRoom temperature or Temperature4 °C until used.



PreparingConcentration0.5 Molarity (M) ReagentNa2EDTASigma Aldrich (100ml)

  • Weigh out Amount18.61 g EDTA disodium salt, dihydrate and add to a 100 mL Duran bottle.
  • Measure outAmount80 mL distilled water and add to the Duran bottle.
  • Add a magnetic flea and place on a magnetic stirring plate to mix the solution. The EDTA salt will not go into solution until thePh8 .
  • Add a pH meter into the solution to observe the pH.
Note
This step may be necessary only when there is EDTA powder available.
For those who have 0.5M EDTA pH 8 they may skip this step and just pipette the required volume of solution to reconstitute.





Weighing the dye powders and reconstituting the concentrated (60x) dye stock
Weighing the dye powders and reconstituting the concentrated (60x) dye stock
10m
10m

Note
This protocol is suited for 10ml concentrated dye stock but can be adjusted for the desired volume.
The concentrated stock is later diluted 10x to give a 6x working stock.

  • With the help of a weighing balance, weigh accuratelyAmount0.015 g ofReagentBromophenol blueSigma Aldrich into a 15ml Eppendorf tube
  • Weigh Amount0.015 g of ReagentXylene cyanolSigma Aldrich into the same tube
  • From a 20%w/v Trehalose stock, aliquot some volume into the Eppendorf tube to dissolve the dye powder.
  • Into the same tube, add Amount120 µL ofConcentration0.5 Molarity (M) EDTA
  • Measure Amount300 µL of Concentration10 % (v/v) SDS into the same tube
  • MeasureAmount2.34 mL of Concentration100 % (v/v) ReagentGlycerolSigma Aldrich and add into the tube
  • Finally add in bits 20% Trehalose to make up the volume to Amount10 mL
  • Cork tightly the Eppendorf tube and mix the content gently to homogenise
  • Label the tube and either store it at room temperature orTemperature4 °C for up to 12 months.
For longer periods, store atTemperature-20 °C




Preparing 6x DNA loading dye (working stock)
Preparing 6x DNA loading dye (working stock)
2m
2m
1:10 Dilution from the concentrated stock

  • From the dye stock, pipette Amount166.6 µL into the Eppendorf tube then add Amount833.3 µL of Concentration20 % (v/v) Trehalose to make a 1ml working solution (6x) of the loading dye. (Or Pipette Amount100 µL from the concentrated dye stock and add Amount900 µL of Concentration20 % (v/v) Trehalose).
  • Cork the tube firmly and mix by inverting the tube several times.
  • Store the working solution at room temperature orTemperature4 °C for up to 12months.
For longer periods, store atTemperature-20 °C



Quality control tests
Quality control tests
The Quality control tests performed for the DNA loading dye include:
  • Functionality test
  • Nuclease test
The protocols can be found here.
Note
We adopted these tests to confirm the integrity and quality of our DNA loading dye formulation but the quality control tests that could be performed are not limited to these two.

Packaging
Packaging
If the DNA loading dye is not used on the spot or is to be sent outside the facility, it is packaged under aseptic conditions to reduce or control contamination that may affect its functionality following the steps in this protocol.
User protocol and trouble shooting
User protocol and trouble shooting
During packaging, a user manual or datasheets are included to help the user effectively use the product. Before using the DNA loading dye, read and follow the instructions described in the user protocol.

If any difficulty or abnormalities are encountered during production or using, refer to the trouble shooting section of the user protocol.