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

这项工作得到了克里斯托弗·纽波特大学 TDG 创业基金的支持。

1. 准备凝胶
注: 关于一般的凝胶电泳协议, 另见 p. y. lee 等。14岁
<ol><li>混合琼脂糖 (~ 1% wv, 特定大小分离的百分比可以变化) 在缓冲液 (约70毫升的小凝胶 (8 x 7 厘米))。缓冲器通常是 TAE (三-醋酸-edta, 40 mM Tris, 20 mm 醋酸, 1 mM EDTA, pH 值约 8.6) 或 TBE (三硼酸盐 edta, 90 mM Tis, 90 mm 硼酸盐, 2 mM EDTA, pH 值约 8.3)。</li>
	<li>将噻唑橙添加到1.3μgml 的最终浓度。<ol>
<li...

<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. 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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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尽管已知毒性, 但溴化乙酯长期以来一直是分子生物学实验室的标准工具。它还受到需要紫外线的影响, 紫外线会在检测到 DNA 时对其造成损害。噻唑橙提供了一种廉价的替代溴化乙酯, 以及有用但昂贵的商业染料。
因此, 噻唑橙的好处是双重的。首先, 噻唑橙可以简单地作为溴化乙酯的替代品。凝胶的制备可以与 EtBr 相同, 用 TO 代替为污渍 (步骤 1.2)。检测极限相似 (~ 1-2 ng/车?...

该方法的目的是利用噻唑橙 (TO) 进行荧光检测, 以确定琼脂糖凝胶中的 DNA。由于其低成本和良好的安全性能, 噻唑橙可能会在从事分子生物学, 特别是结扎和克隆的本科教学实验室和研究实验室中看到特别的好处。
溴化乙酯仍然是检测琼脂糖凝胶中 DNA 的最常见染料。这主要是因为它可以非常便宜地获得, 只需要用紫外线进行激发进行检测。溴化乙酯和噻唑橙价格低廉, 检出限低 (1-2 ng/lane)1。有两个主要的缺点, 乙二酸溴, 然而, 噻唑橙改善。
首先, 溴化乙酯是一种具有特殊处理、运输和处置要求的突变体2 , 而噻唑橙剂的致突变性较低 (在艾姆斯试验中3–4x 较少致突变性)3、4, 一般可以用常见的化学废物。
其次, 溴化物需要紫外线进行检测。如果需要, 噻唑橙同样可以使用紫外线, 但也可以用蓝光检测。紫外线虽然使用广泛, 但也有一些明显的缺点。首先, 它损害了人体的皮肤和眼睛。虽然紫外线可以由训练有素的专业人员安全使用, 但实验室紫外线对皮肤或眼睛的意外损害 (功能类似于晒伤) 并不少见, 尤其是在经验不足的科学家中。其次, 紫外线对 dna 样本5的危害极大, 这降低了下游实验 (如结扎和转化)1,6,7的成功。允许用蓝光进行探测 (ex, max = 510 nm (488 nm 和 470 nm 也显示出强烈的激发)), 不会造成皮肤损伤或 dna 损伤 (尽管任何强光可能仍然对眼睛有害), 大大降低了科学家面临的风险和样品。
TO 并不是溴化乙酯的唯一荧光染料替代品;它的优势在于成本。to 在20世纪80年代被发现为网状细胞染色8, 并已在一些基于 dna 的荧光实验9,10,11, 12,13中发现了有用的。它目前由多个供应商出售。TO 是额外的、更昂贵的蓝光--可检测到的商业染料的父化合物, 在电泳过程中表现类似, 使用紫外线或蓝光进行检测1。此外, 虽然其他染料对 Dna 浓度比 EtBr 或 TO 更敏感, 但对于一般电泳实验, 此类染料在许多情况下的成本高得令人望而却步。

<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

使用琼脂糖进行 DNA 凝胶电泳是分子生物学实验室中常见的工具, 可以按大小分离 DNA 片段。分离后, DNA 通过染色进行可视化。这篇文章演示如何使用噻唑橙染色 DNA。与普通染色方法相比, 噻唑橙与普通染色方法相比是有利的, 因为它敏感、价格低廉、可与紫外线或蓝光激发 (以防止样品损坏), 并且比溴化乙酯更安全。已经配备了使用溴化乙酯进行 DNA 电泳实验的实验室通常可以在不对现有协议作额外修改的情况下切换染料, 使用紫外线进行检测。蓝光检测, 以避免样品损坏, 还可以实现与蓝光源和发射过滤器。已经配备了蓝光检测设备的实验室可以简单地切换染料, 而无需对现有协议进行其他更改。

噻唑橙可以检测 DNA, 无需使用溴化乙酯, 也无需使用 dna 有害的紫外线。溴化乙酯是众所周知的诱变性, 因此从实验室中消除它可能是有利的。紫外线会显著损害 DNA 并降低转化效率, 而蓝光不会对 DNA 造成损害。溴化乙酯、噻唑橙和一种普通的蓝光--可检测的商业 DNA 染料 (图 1, 见材料表) 之间的检测限值相似, 所有三种染料的检测限值均为微型凝胶中的 ~ 1-2 ng/lane<...

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

用噻唑橙代替溴代或替代染料进行 DNA 电泳

在这里, 我们提出了一个协议, 使用噻唑橙检测 DNA 的凝胶电泳实验。使用噻唑橙可以消除溴化乙酯, 并且可以使用紫外或蓝光进行荧光检测。

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, ...

该协议是使用溴化乙基在电泳期间进行DNA检测的一种简单而廉价的替代方案。通过去除溴化乙基，可以使用紫外线或蓝光进行检测。蓝光不会损害DNA，这提高了下游实验成功的机会。尝试这个真的很容易。你只需要用硫酮橙代替凝胶中的溴化乙酰胺。首先，在70毫升三醋酸-EDTA或三盐-玻酸缓冲液中混合约0.7克阿加罗斯，准备1%的迷你凝胶。接下来，在 DMSO 中溶解硫酮橙，使 10，000X 库存溶液。虽然不是特别对光敏感，但不使用时，在黑暗中将硫酮橙色储存。对于长期储存，制作硫松橙-DMSO混合物的等分，并冷冻。然后，将硫酮橙加入每毫升1.3微克的最终浓度，微波将阿加罗斯缓冲液和硫酮橙的混合物溶解约60秒。将阿加罗斯混合物倒入含有适当梳子的凝胶铸造装置中，使加糖溶液凝固成凝胶。要加载凝胶，请先将凝胶放在电泳装置中（如果不存在）。然后添加 TAE 或 TBE 运行缓冲液以覆盖凝胶表面。接下来，使用加载染料，加载10微升的DNA样本。包括DNA尺寸梯供参考。连接盖和电极，以 100 伏特运行迷你凝胶，直到加载染料在迷你凝胶中移动约 4 到 7 厘米。如果在凝胶铸造之前未应用硫佐尔橙，则将凝胶浸入含有硫酮橙的缓冲液中，轻轻搅拌约 20 分钟，或直到完全检测到带子。要使用紫外线透光器对凝胶进行可视化，请从电泳设备中去除凝胶，并将其放在紫外线透光器上。要使用蓝光透光器或手电筒对凝胶进行可视化，请将凝胶放在蓝光转光器上，或从上方或下方将蓝色 LED 手电筒直接放在凝胶上。使用琥珀色发射过滤器过滤出蓝光，实现脱氧核糖核酸橙色复合物的荧光可视化，并保护染料。为了进一步消化或结扎，从凝胶中切出所需的DNA带，然后使用现成的试剂盒或协议提取DNA。在凝胶成像设备中选择适当的激发和发射设置。在没有带适当滤光片的成像系统的情况下，在摄像机和凝胶蓝光激发源之间放置琥珀色滤光片。溴化乙基、硫酮橙和常见的蓝光可检测商业DNA染料之间的检测限值相似，所有三种染料的检测限值在迷你凝胶中每通道一至两个纳米。当使用紫外线或蓝光透光器或蓝光手电筒进行兴奋时，可检测到限制消化的硫橙染色的阿加罗斯凝胶。虽然橙色硫化物似乎比溴化乙基危险性小，但仍应佩戴标准的个人防护设备，如手套和护目镜。

Research

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Biochemistry

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Sheathless Capillary ElectrophoresisÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œMass Spectrometry for Metabolic Profiling of Biological Samples

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科研

生物化学

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Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

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Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

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For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C

用酮抑制剂 PD0325901 和维生素 C 维持小鼠胚胎干细胞基因组低甲基化的一种替代培养方法

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many ...

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

二维半变性洗涤剂琼脂糖凝胶电泳法确定淀粉样或淀粉样纤维的粒度异质性

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

毛细管电泳与亲和毛细管电泳对金属离子 AtHIRD11 固有紊乱与结合的分析

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

蓝色本机凝胶电泳法分析囊体膜蛋白配合物

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA序列识别利用高通量原酶分析的DNAPrimase

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...