TITLE:  Production of 10000x  TO-DMSO DNA gel stain
AUTHORS: Shalo  Minette, Stephane  Fadanka, Nadine    Mowoh

[DESCRIPTION]
DNA dyes stain deoxyribonucleic acid for laboratory purposes such as detection and quantification. Many DNA dyes also bind to RNA and could be more broadly described as nucleic acid stains. Common dyes included ethidium bromide (EtBr), especially for agarose gel electrophoresis of DNA.

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, DNA is visualized by staining.

Thiazole orange compares favorably to common staining methods in that it is sensitive, inexpensive, excitable with UV or blue light (to prevent sample damage), and safer than ethidium bromide.
Thiazole orange is known to interact with DNA and produce significant fluorescence which can be used to visualize DNA in electrophoresis gels.
Thiazole orange powder can dissolve completely without precipitating in several solvents except water.
 
Scope:

This protocol covers the steps involved in making a 10000x stock DNA gel stain using Thiazole orange dye powder.
This protocol also covers the steps involved in using the 10000x stock to prepare a 1.3ug/ml final concentration of gel stain required to visualize DNA bands on an agarose gel.

The solvent used for this protocol is DMSO.

[BEFORE_START]
Make sure to assemble and keep ready all materials, equipment and reagents needed for the procedure.
Check to be sure the weighing balance and Micro pipette is working correctly (are able to weigh the right mass in the right units, and picks up the right volume respectively).

[GUIDELINES]
It is advisable to prepare the DNA gel stain in large batches(10-15 tubes) to enable easy weighing of the powder especially in situations where a high precision balance is unavailable.

[STEPS]
SECTION: Preparing a 13mg/ml stock of DNA gel stain (1ml)
1. Use a weighing balance to carefully measure0.013 g of   into a 1.5ml Eppendorf tube or opaque screw cap tube.

SECTION: Preparing a 13mg/ml stock of DNA gel stain (1ml)
2. Use a micropipette to pipette 1000 µL of   into the same Eppendorf tube.

SECTION: Preparing a 13mg/ml stock of DNA gel stain (1ml)
3. Mix by gently inverting the tube several times to have a 10000x stock. This step may take a minute or less. 1 min

For immediate or daily use, leave at room temperature away from contact with sunlight (It can be stable up to 3 months. If not immediately used, store the gel stain stock in the fridge or freeze for 6 to 12 months.

 

 

SECTION: Preparing a 1.3µg/ml final concentration of DNA gel stain for agarose gel electrophoresis
4. Follow this step to prepare a 1X TBE buffer from a 10X stock

Prepare a 2% agarose gel by diluting 0.5g of agarose powder in 25ml 1x TBE or (calculate the corresponding amount of agarose to use for the  percentage and volume of agarose gel desired).

From the 10000x DNA gel stain stock, pipette2.5 µLinto 25 mL molten agar

Cast the gel and leave to polymerize on the working bench atRoom temperature. This may take about20 min.

Load and run the gel immediately or you can keep the gel on the bench at room temperature till you are ready ( the gel can stay up to 3 to 6 hours without affecting visualization of DNA bands).
SECTION: Quality control tests  for TO-DMSO gel stain
5. The following quality control tests are performed after producing the 10000x DNA gel stain to confirm it meets its desired specifications.



SECTION: Functionality test
6. 
 

SECTION: Functionality test
7. Amplification of DNA template in a PCR reaction

Thaw all reagents on ice in a bowl

Label reaction tubes (PCR tubes) according to the number of samples, and including controls in each run (negative and positive controls) as needed.

If using a 10x cellular reagent enzyme- Rehydrate the enzyme with 30μl of sterile PCR grade water, flick tube and keep on ice.

 

SECTION: Functionality test
8. In each PCR tube, pipette and combine the following reagent components following the order in the table below (work on ice while pipetting)

 

SECTION: Functionality test
9. Gently mix the reaction tubes by flicking and transfer in the PCR machine to begin thermocycling.
SECTION: Functionality test
10. Connect the thermocycler and program the protocol to run following the thermocycling steps listed below. The exact annealing temperature will depend on the choice of DNA template and primers used.

 

SECTION: Functionality test
11. 
 

SECTION: Functionality test
12. Preparing and running agarose gel electrophoresis

About 30 min to the end of the thermocycling process, prepare a 1x TBE buffer from 10x stock following this protocol and use it to make a 2% agarose gel following this protocol.
 



SECTION: Functionality test
13. Loading the agarose gel wells

Pipette 3 to 5μl of DNA ladder (size depending on size of the DNA template) unto the first well then pipette the negative control carefully to avoid bubbles and load into the second well.

Following the step as described  here, mix your amplicons with DNA loading dye and load in the subsequent wells

Allow the gel to run for 15-30mins at 48V on 0.8 or 2% agarose gel and TBE buffer
 
SECTION: Functionality test
14. Visualization

The amplification results are visualized by transferring the gel into a UV transilluminator or blue gel transilluminator

With the help of the DNA ladder, determine if the amplification product is of the expected size and the bands are sharp and distinct looking exactly like those from the EtBr gel to show that the TO-DMSO gel stain was able to stain the DNA. 
SECTION: Confirm visualization of low molecular weight DNA
15. 
 
Follow the steps in preparing a 2% agarose gel and load as described in step 17 above. DNA templates or amplicon in this case  is a 100bp DNA ladder.
Run gel to finish and visualize the resulting band separation to confirm separation of the ladder into visible distinct bands .
SECTION: Confirmation of visualization of low concentration of DNA
16. 
 

Make 1:5 serial dilutions of the 100bp DNA ladder or any DNA amplicon and use the different dilutions as amplicons to load a gel well.
Allow the gel to run through and visualize to determine the lowest concentration detectable by the gel stain.

At this stage, the gel stain is ready to be used either on the spot or can be shipped or sent to an external lab and used following the steps described in the user protocol or instructions in step 4 above.