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

Summary

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.

Abstract

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.

Introduction

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

Protocol

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)....

Results

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 Materials<.......

Discussion

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.......

Materials

List of materials used in this article

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