TITLE: Production of 6x DNA Loading Dye
AUTHORS: Stephane  Fadanka, Nadine    Mowoh

[DESCRIPTION]
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. 


[BEFORE_START]
Check to make sure you have all the starting materials available and assembled to prepare the reagent stocks.

[STEPS]
SECTION: Preparation of Reagent stocks
1. 
 


Preparing 20 % (v/v)Trehalose (100ml)

Accurately weigh 20 g of   powder into a 500 mLbeaker
Use a measuring cylinder to measure100 mLof 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 4 °C until used.


SECTION: Preparation of Reagent stocks
2. Preparing 10 % (v/v)  (100ml)

Accurately weigh 10 g of SDS powder into a 500 mL beaker
Use a measuring cylinder to measure 100 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 Room temperature  or 4 °C until used.



SECTION: Preparation of Reagent stocks
3. Preparing0.5 Molarity (M)  (100ml) 

Weigh out 18.61 g EDTA disodium salt, dihydrate and add to a 100 mL Duran bottle.
Measure out80 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 thepH 8.
Add a pH meter into the solution to observe the pH.
 





SECTION: Weighing the dye powders and reconstituting the concentrated (60x) dye stock
4. 
 

With the help of a weighing balance, weigh accurately0.015 g of  into a 15ml Eppendorf tube
Weigh 0.015 g of   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 120 µL of0.5 Molarity (M)EDTA
Measure 300 µL of 10 % (v/v)SDS into the same tube
Measure2.34 mL of 100 % (v/v) and add into the tube
Finally add in bits 20% Trehalose to make up the volume to 10 mL
Cork tightly the Eppendorf tube and mix the content gently to homogenise
Label the tube and either store it at room temperature or4 °C for up to 12 months.
For longer periods, store at-20 °C




SECTION: Preparing 6x DNA loading dye (working stock)
5. 1:10 Dilution from the concentrated stock

From the dye stock, pipette 166.6 µLinto the Eppendorf tube then add 833.3 µLof 20 % (v/v)Trehalose to make a 1ml working solution (6x) of the loading dye. (Or Pipette 100 µL from the concentrated dye stock and add 900 µL of 20 % (v/v)Trehalose).
Cork the tube firmly and mix by inverting the tube several times.
Store the working solution at room temperature or4 °C for up to 12months.
For longer periods, store at-20 °C



SECTION: Quality control tests
6. The Quality control tests performed for the DNA loading dye include:
Functionality test
Nuclease test
The protocols can be found here.
 

SECTION: Packaging
7. 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.
SECTION: User protocol and trouble shooting
8. 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.