Name: dna electrophoresis using thiazole orange instead of ethidium bromide or alternative dyes
Uploaded: 2019-03-31T13:00:00
Description: Watch this Scientific Journal Video about DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes at JoVE.com

This work was supported by startup funds to TDG from Christopher Newport University.&#160;

1. Preparing the gel
NOTE: For general gel electrophoresis protocols, see also P.Y. Lee, et al.14.&#160;
<ol>
	<li>Mix agarose (~1% w/v, percentage can be varied for particular size separations) in buffer (approximately 70 mL for a mini-gel (8 x&#160;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)).</li>
	<li>Ad...

<ol>
	<li>O'Neil, C. S., Beach, J. L., Gruber, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiazole+orange+as+an+everyday+replacement+for+ethidium+bromide+and+costly+DNA+dyes+for+electrophoresis.">Thiazole orange as an everyday replacement for ethidium bromide and costly DNA dyes for electrophoresis.</a> Electrophoresis. 39 (12), 1474-1477 (2018).</li><li>McCann, J., Choi, E., Yamasaki, E., Ames, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+carcinogens+as+mutagens+in+the+Salmonella/microsome+test:+assay+of+300+chemicals.">Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals.</a> Proceedings of the National Academy of Sciences of the United States of America. 72 (12), 5135-5139 (1975).</li><li>Evenson, W. E., Boden, L. M., Muzikar, K. A., O&#8217;Leary, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1H+and+13C+NMR+Assignments+for+the+Cyanine+Dyes+SYBR+Safe+and+Thiazole+Orange.">1H and 13C NMR Assignments for the Cyanine Dyes SYBR Safe and Thiazole Orange.</a> The Journal of Organic Chemistry. 77 (23), 10967-10971 (2012).</li><li>Beaudet, M., Cox, G., Yue, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Probes, Inc. , (2005).</li><li>Pfeifer, G. P., You, Y. H., Besaratinia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+induced+by+ultraviolet+light.">Mutations induced by ultraviolet light.</a> Mutation Research. 571 (1-2), 19-31 (2005).</li><li>Cariello, N. F., Keohavong, P., Sanderson, B. J., Thilly, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+produced+by+ethidium+bromide+staining+and+exposure+to+ultraviolet+light.">DNA damage produced by ethidium bromide staining and exposure to ultraviolet light.</a> Nucleic Acids Research. 16 (9), 4157 (1988).</li><li>Hartman, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transillumination+can+profoundly+reduce+transformation+frequencies.">Transillumination can profoundly reduce transformation frequencies.</a> BioTechniques. 11 (6), 747-748 (1991).</li><li>Lee, L. G., Chen, C. H., Chiu, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiazole+orange:+a+new+dye+for+reticulocyte+analysis.">Thiazole orange: a new dye for reticulocyte analysis.</a> Cytometry. 7 (6), 508-517 (1986).</li><li>Nygren, J., Svanvik, N., Kubista, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Interactions+Between+the+Fluorescent+Dye+Thiazole+Orange+and+DNA.">The Interactions Between the Fluorescent Dye Thiazole Orange and DNA.</a> Biopolymers. , 1-13 (1998).</li><li>Svanvik, N., Westman, G., Wang, D., Kubista, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-Up+Probes:+Thiazole+Orange-Conjugated+Peptide+Nucleic+Acid+for+Detection+of+Target+Nucleic+Acid+in+Homogeneous+Solution.">Light-Up Probes: Thiazole Orange-Conjugated Peptide Nucleic Acid for Detection of Target Nucleic Acid in Homogeneous Solution.</a> Analytical Biochemistry. 281 (1), 26-35 (2000).</li><li>Yang, P., De Cian, A., Teulade-Fichou, M. P., Mergny, J. L., Monchaud, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+Bisquinolinium/Thiazole+Orange+Conjugates+for+Fluorescent+Sensing+of+G-Quadruplex+DNA.">Engineering Bisquinolinium/Thiazole Orange Conjugates for Fluorescent Sensing of G-Quadruplex DNA.</a> Angewandte Chemie International Edition. 48 (12), 2188-2191 (2009).</li><li>Fang, G. M., Chamiolo, J., Kankowski, S., Hovelmann, F., Friedrich, D., Lower, A., Meier, J. 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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.&#160;
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...

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. There are two main drawbacks to ethidium bromide, however, that thiazole orange improves upon.&#160;
First, ethidium bromide is a mutagen2 with special handling, shipping, and disposal requirements, whereas thiazole orange is less mutagenic (3&#8211;4x less mutagenic in Ames test)3,4 and can be generally disposed of with common chemical waste.
Second, ethidium bromide requires UV light for detection. Thiazole orange can similarly use UV light if desired, but can also be detected with blue light. UV light, while commonly used, has a few salient disadvantages. First, it is damaging to human skin and eyes. While UV light can be used safely by trained professionals, accidental skin or eye damage (functionally similar to sunburns) from laboratory UV light are not uncommon particularly with inexperienced scientists. Second, UV light is extremely damaging to DNA samples5, which reduces the success of downstream experiments (such as ligation and transformation)1,6,7. TO allows detection with blue light (&#955;ex,max = 510 nm (488 nm and 470 nm also show strong excitation)), which does not cause skin damage or DNA damage (although any intense light may still be harmful to eyes), greatly decreasing the risks to both the scientist and the sample.&#160;
TO is not the only fluorescent dye alternative to ethidium bromide; its advantage is cost. TO was discovered in the 1980s as a reticulocyte stain8, and has found utility in a number of DNA-based fluorescence experiments9,10,11,12,13. It is currently sold by multiple suppliers. TO is the parent compound of additional, more expensive, blue-light&#8211;detectable commercial dyes, and behaves similarly during electrophoresis, using UV or blue light for detection1. Furthermore, while other dyes are more sensitive to very low DNA concentrations than either EtBr or TO, for generic electrophoresis experiments, such dyes are prohibitively expensive in many contexts.&#160;

<table border="0" cellpadding="0" cellspacing="0" style="width:572px;" width="573">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2-log DNA ladder</td>
			<td style="width:71px;">New England Biolabs</td>
			<td style="width:119px;">N0469S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Agarose (Genetic Analysis Grade)</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP1356-100</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Blue-light flashlight</td>
			<td style="width:71px;">WAYLLSHINE (Amazon)</td>
			<td style="width:119px;">WAYLLSHINE Scalable Blue LED</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ChemiDoc MP</td>
			<td style="width:71px;">Biorad</td>
			<td style="width:119px;">1708280</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMSO</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ethidium bromide</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP1302-10</td>
			<td>For comparison, not necessary for protocol</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gel apparatus (Owl Easy Cast)</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">B1A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Qiagen Qiaquick Gel extraction kit</td>
			<td style="width:71px;">Qiagen</td>
			<td style="width:119px;">28704</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Safe Imager Viewing Glasses</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">S37103</td>
			<td>Necessary for using blue light flashlight.*</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SafeImager 2.0 (Blue light transilluminator)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">G6600</td>
			<td>Blue light flashlight may be used as alternative</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SYBR Safe</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">S33102</td>
			<td>For comparison, not necessary for protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TAE (Tris-Acetate-EDTA)</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">46-010-CM</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thiazole orange</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">390062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>*Glasses are also included with Invitrogen G6600</td>
		</tr>
	</tbody>
</table>

dna electrophoresis using thiazole orange instead of ethidium bromide or alternative dyes

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.

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&#8211;detectable commercial DNA dye (Figure 1, see Table of Mate...

Watch this Scientific Journal Video about DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes at JoVE.com

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

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, ...

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