DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

1. Preparing the gel

NOTE: For general gel electrophoresis protocols, see also P.Y. Lee, et al.¹⁴. 

1. Mix agarose (~1% w/v, percentage can be varied for particular size separations) in buffer (approximately 70 mL for a mini-gel (8 x 7 cm)). Buffers are commonly TAE (tris-acetate-EDTA, 40 mM Tris, 20 mM acetate, 1 mM EDTA, pH approximately 8.6) or TBE (tris-borate-EDTA, 90 mM Tris, 90 mM borate, 2 mM EDTA, pH approximately 8.3)).
2. Add thiazole orange to a final concentration of 1.3 μg/mL.
   1. Dissolve thiazole orange in DMSO to make a 10,000x stock solution (13 mg/mL). While not particularly light sensitive, store TO in the dark when not in use. This solution is stable at room temperature for ~months; long-term storage may be achieved by freezing aliquots (DMSO will freeze in a standard refrigerator). Be sure to entirely melt and resuspend a frozen solution before use. 
      NOTE: The gel can also be stained with TO after electrophoresis (see step 2.6). While TO has an improved safety profile over ethidium bromide, standard molecular biology laboratory safety precautions should be maintained. 
3. Microwave the mixture of agarose, buffer, and thiazole orange to dissolve agarose (approximately 60 s). This step is commonly referred to as “melting”. Swirl (5 s) to aid dissolution if needed. 
   NOTE: TO can also be added after microwaving if preferred.
4. Allow the agarose solution to cool briefly before pouring into gel casting apparatus containing an appropriate comb.
5. Allow the agarose solution to solidify into a gel. 

2. Loading and running the gel

1. Place the gel in the electrophoresis apparatus if not already present.
2. Add running buffer (TAE or TBE as above) to cover the surface of the gel.
3. Load DNA samples (commonly 10 μL) using a loading dye. Include a DNA sizing ladder for reference.
4. Attach the cover and electrodes (the gel should be run toward the red anode (positive)).
5. Apply voltage (typically ~100V for a mini-gel, although size of gel may require a modified voltage to prevent damaging the gel) until loading dye has traveled an appropriate distance (approximately 4-7 cm for a mini-gel, although distance may vary depending on precise application).
   NOTE: Like ethidium bromide and many other DNA-binding dyes, thiazole orange is positively charged. Consequently, these dyes will migrate in the opposite direction of electrophoresing DNA. For samples which are run far down the gel for enhanced separation, eventually the dye will separate from the smaller DNA fragments, resulting in weak staining. In these instances, the gel should be stained as in step 2.6. This situation is not unique to TO, any positively charged dye that reversibly interacts with DNA will exhibit this behavior (including ethidium bromide, TO, and others).
6. If TO was not added prior to gel casting (step 1.2), stain by immersing the gel in the buffer containing thiazole orange.
   1. Prepare enough buffer (TAE or TBE) containing 1.3 μg/mL thiazole orange to completely cover the gel and soak the gel with gentle agitation until the bands are fully detected (roughly 20 min).

3. Visualization of thiazole orange agarose gel (UV transilluminator)

1. Remove the gel from the electrophoresis apparatus and place on a UV transilluminator.
   CAUTION: UV light is damaging to skin and eyes. Be sure to wear appropriate eye (goggles) and face (face shield) protection. Hands should have gloves and long sleeves should be worn.
2. If desired, cut out desired DNA bands from the gel (for further digestion or ligation, for example). Expose the gel to UV light for as short a length of time as possible during excision. UV light (regardless of DNA dye) damages DNA. 
3. Extract DNA from the gel slice using a readily available kit or protocol¹⁵.

4. Visualization of thiazole orange agarose gel (blue-light transilluminator or flashlight)

1. Remove the gel from the electrophoresis apparatus and place on a blue-light transilluminator (~470 nm maximum emission wavelength). Alternatively, a blue LED (~470 nm) flashlight can be directed at the gel (either from above or below).
   NOTE: While sensitivity is lower using a blue-light transilluminator with ethidium bromide, DNA stained with ethidium bromide can be detected using this blue-light protocol.
2. Use an amber emission filter (~560 nm longpass, either goggles or square) to filter blue light, enabling visualization of fluorescence from DNA:thiazole orange complexes. 
   NOTE: Without the amber emission filter, it is very difficult to detect DNA bands due to intensity of blue-light excitation source. 
   CAUTION: Although blue light lacks the ability to acutely damage tissues (contrasting UV), prolonged exposure to intense blue light could damage eyes and amber emission goggles or filter should be used.
3. If desired, cut out desired DNA bands from the gel for further applications.
   NOTE: Since blue light does not damage DNA, it is not necessary to rapidly cut out bands (such as when using UV excitation in step 3.2).
4. Extract DNA from the gel slice using a readily available kit or protocol¹⁵.

5. Image capture

1. Select appropriate excitation and emission settings in gel-imaging apparatus. The excitation and emission of thiazole orange (λ_ex,max = 510 nm (488 nm and 470 nm also show strong excitation, in addition to strong excitation at UV wavelengths); λ_em = 527 nm) are nearly identical to common blue-light–detectable commercial dyes, so instruments may have preset filter settings that can be used. 
   NOTE: Some filter settings for blue-light–detectable commercial dyes actually use damaging UV light for excitation, so use caution if imaging prior to cutting out bands. Use a blue-light excitation source if possible when DNA bands will be excised after imaging.
2. In the absence of an imaging system with appropriate filters, place an amber filter between the camera and the gel/blue-light excitation source.