This protocol is an easy and inexpensive alternative to using ethidium bromide for DNA detection during electrophoresis. By removing ethidium bromide, either UV light or blue light can be used for detection. Blue light is not damaging to DNA, which improves the chance of success for downstream experiments.
It's really easy to try this out. You just have to replace the ethidium bromide in your gels with thiazole orange. To begin, prepare 1%mini-gels by mixing approximately 0.7 grams of agarose in 70 milliliters of tris-acetate-EDTA or tris-borate buffers.
Next, dissolve thiazole orange in DMSO to make a 10, 000X stock solution. While not particularly light sensitive, store thiazole orange in the dark when not in use. For long-term storage, make aliquots of the thiazole orange-DMSO mixture and freeze them.
Then, add thiazole orange to a final concentration of 1.3 micrograms per milliliter, and microwave the mixture of agarose buffer and thiazole orange to dissolve agarose for approximately 60 seconds. Pour the agarose mixture into a gel casting apparatus containing an appropriate comb, and allow the agarose solution to solidify into a gel. To load the gel, first place the gel in the electrophoresis apparatus, if not already present.
Then add TAE or TBE running buffer to cover the surface of the gel. Next, using a loading dye, load 10 microliters of a DNA sample. Include a DNA sizing ladder for reference.
Attach the cover and electrodes, and run the mini-gel at 100 volts until the loading dye travels approximately four to seven centimeters for a mini-gel. If thiazole orange was not applied prior to gel casting, stain the gel by immersing it in the buffer containing thiazole orange with gentle agitation for about 20 minutes or until the bands are fully detected. To visualize the gel using a UV transilluminator, remove the gel from the electrophoresis apparatus and place it on the UV transilluminator.
To visualize the gel using a blue light transilluminator or a flashlight, place the gel on the blue light transilluminator or direct the blue LED flashlight at the gel from above or below. Use an amber emission filter to filter out the blue light, enabling visualization of fluorescence from DNA thiazole orange complexes and to protect dyes. For further digestion or ligation, cut out desired DNA bands from the gel and proceed to extracting DNA using a readily available kit or protocol.
Select appropriate excitation and emission settings in the gel-imaging apparatus. In the absence of an imaging system with appropriate filters, place an amber filter between the camera and the gel blue-light excitation source. Detection limits are similar between ethidium bromide, thiazole orange, and a common blue-light-detectable commercial DNA dye, with the detection limit for all three dyes being one to two nanograms per lane in a mini-gel.
Thiazole orange-stained agarose gel of a restriction digest is detectable when it is excited with a UV or a blue light transilluminator or with a blue light flashlight. While thiazole orange appears to be less hazardous than ethidium bromide, standard personal protective equipment such as gloves and goggles should still be worn.
