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Here, we present a protocol to use thiazole orange for the detection of DNA in gel electrophoresis experiments. The use of thiazole orange allows elimination of ethidium bromide, and fluorescence detection can be achieved with either UV or blue light.
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. This article demonstrates how to use thiazole orange to stain DNA. 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. Labs already equipped to run DNA electrophoresis experiments using ethidium bromide can generally switch dyes with no additional changes to existing protocols, using UV light for detection. Blue-light detection to avoid sample damage can additionally be achieved with a blue-light source and emission filter. Labs already equipped for blue-light detection can simply switch dyes with no additional changes to existing protocols.
The purpose of this method is to identify DNA in agarose gels using thiazole orange (TO) for fluorescence detection. Due to its low cost and favorable safety profile, thiazole orange may see particular benefit in undergraduate teaching labs and research labs performing molecular biology, especially ligations and cloning.
Ethidium bromide remains the most common dye for detection of DNA in agarose gels. This is primarily because it can be obtained very inexpensively and only requires excitation with UV light for detection. Both ethidium bromide and thiazole orange are inexpensive, with low detection limits (1-2 ng/lane)1
1. Preparing the gel
NOTE: For general gel electrophoresis protocols, see also P.Y. Lee, et al.14.
Thiazole orange enables detection of DNA, without using ethidium bromide and without using DNA-damaging UV light. Ethidium bromide is well-known to be mutagenic, so eliminating it from the lab may be advantageous. UV light damages DNA and lowers transformation efficiency significantly, whereas blue light does not damage DNA. Detection limits are similar between ethidium bromide, thiazole orange, and a common, blue-light–detectable commercial DNA dye (Figure 1, see Table of Mate.......
Ethidium bromide has long been a standard tool in the molecular biology lab, despite known toxicity. It also suffers from requiring UV light, which damages the DNA as it is being detected. Thiazole orange offers an inexpensive alternative to ethidium bromide, as well as useful but expensive commercial dyes.
The benefits of thiazole orange are thus two-fold. First, thiazole orange can simply be used as a replacement to ethidium bromide. Gels can be prepared identically to EtBr, with TO su.......
This work was supported by startup funds to TDG from Christopher Newport University.
....Name | Company | Catalog Number | Comments |
2-log DNA ladder | New England Biolabs | N0469S | |
Agarose (Genetic Analysis Grade) | Fisher | BP1356-100 | |
Blue-light flashlight | WAYLLSHINE (Amazon) | WAYLLSHINE Scalable Blue LED | |
ChemiDoc MP | Biorad | 1708280 | |
DMSO | Sigma-Aldrich | D8418 | |
ethidium bromide | Fisher | BP1302-10 | For comparison, not necessary for protocol |
Gel apparatus (Owl Easy Cast) | Thermo Scientific | B1A | |
Qiagen Qiaquick Gel extraction kit | Qiagen | 28704 | |
Safe Imager Viewing Glasses | Invitrogen | S37103 | Necessary for using blue light flashlight.* |
SafeImager 2.0 (Blue light transilluminator) | Invitrogen | G6600 | Blue light flashlight may be used as alternative |
SYBR Safe | Invitrogen | S33102 | For comparison, not necessary for protocol |
TAE (Tris-Acetate-EDTA) | Corning | 46-010-CM | |
Thiazole orange | Sigma-Aldrich | 390062 | |
*Glasses are also included with Invitrogen G6600 |
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