Name: dna staining method based on formazan precipitation induced by blue light exposure
Uploaded: 2018-01-28T14:00:00
Description: Watch this Scientific Journal Video about DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure at JoVE.com

这项工作得到了基金发展 Científico y Tecnológico (科技), 智利, 项目 11130263 (cw), 项目会 + NERC + 方案 de Colaboración 国际 PCI-PII20150073 (对 cw) 和 U inicia 从 Vicerrectoría de智利研究大学 (对 CW)。
感谢纳兰霍-帕尔马在这个项目的初始阶段的帮助, Dr. 巴布尔和迭基罗加-罗杰为有益的讨论, 罗伯特 Lesch 修订手稿和总署 de extensión Facultad 德 Ciencias Químicas yFarmacéuticas 来自智利大学。也感谢马塞洛佩雷斯的编辑视频和尼尔史蒂文斯为纠正文本和沃尔华提供的声音-结束。

1. 凝胶的制备和运行
<ol><li>用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 (sds 页) 在理查德·山姆布鲁克和罗素14中发现的不变性12% 聚丙烯酰胺凝胶的配方, 但使用水代替 sds。<ol>
<li>要准备5毫升的溶解凝胶, 使用1.7 毫升蒸馏水, 2 毫升丙烯酰胺/双丙烯酰胺 (30/0.8% 瓦特/五), 1.3 毫升1.5 米的 pH 8.8, 0.05 毫升的10% 硫酸铵, 和0.002 毫升的 TEMED。</li>
		<li>准备2毫升的堆叠凝胶使用1.5 毫升水, 0.33 ...

<ol>
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E., 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=Comparison+of+SYBR+Green+I+nucleic+acid+gel+stain+mutagenicity+and+ethidium+bromide+mutagenicity+in+the+Salmonella/mammalian+microsome+reverse+mutation+assay+(Ames+test).">Comparison of SYBR Green I nucleic acid gel stain mutagenicity and ethidium bromide mutagenicity in the Salmonella/mammalian microsome reverse mutation assay (Ames test).</a> Mutat Res. 439 (1), 37-47 (1999).</li><li>Xin, X., Yue, S., Wells, K. S., Singer, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SYBR+Green-I:+a+new+fluorescent+dye+optimized+for+detection+picogram+amounts+of+DNA+in+gels.">SYBR Green-I: a new fluorescent dye optimized for detection picogram amounts of DNA in gels.</a> Biophys. 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I., et al. <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+DNA+using+a+visible+dye,+Nile+blue,+in+electrophoresed+gels.">Detection of DNA using a visible dye, Nile blue, in electrophoresed gels.</a> Anal. Biochem. 280 (2), 322-324 (2000).</li><li>Santill&#225;n-Torres, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+stain+for+DNA+in+agarose+gels.">A novel stain for DNA in agarose gels.</a> Trends Genet. 9 (2), 40 (1993).</li><li>Adkins, S., Burmeister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+DNA+in+agarose+gels+as+migrating+colored+bands:+applications+for+preparative+gels+and+educational+demonstrations.">Visualization of DNA in agarose gels as migrating colored bands: applications for preparative gels and educational demonstrations.</a> Anal. Biochem. 240 (1), 17-23 (1996).</li><li>Cong, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+visible+dye-based+staining+method+for+DNA+in+polyacrylamide+gels+by+ethyl+violet.">A visible dye-based staining method for DNA in polyacrylamide gels by ethyl violet.</a> Anal. Biochem. 402 (1), 99-101 (2010).</li><li>Altman, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrazolium+salts+and+formazans.">Tetrazolium salts and formazans.</a> Prog. Histochem. Cytochem. 9 (3), 1-56 (1976).</li><li>Nineham, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemistry+of+formazans+and+tetrazolium+salts.">The chemistry of formazans and tetrazolium salts.</a> Chem. Rev. 55 (2), 355-483 (1955).</li><li>Crandall, I. E., Zhao, B., Vlahakis, J. Z., Szarek, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+imidazole-,+imidazolium,+and+tetrazolium-containing+compounds+with+DNA.">The interaction of imidazole-, imidazolium, and tetrazolium-containing compounds with DNA.</a> Bioorg. Med. Chem. Lett. 23 (5), 1522-1528 (2013).</li><li>Paredes, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+visible+and+selective+DNA+staining+method+in+gels+with+tetrazolium+salts.">New visible and selective DNA staining method in gels with tetrazolium salts.</a> Anal. Biochem. 517, 31-35 (2017).</li><li>Green, M. R., Sambrook, J. <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 Cloning: a Laboratory Manual. , (2012).</li><li>Hansen, W., Garcia, P. 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本文介绍了一种灵敏、新颖的基于 NBT 还原的 DNA 染色方法及其在 SG I 中的应用。该协议的关键步骤是凝胶在 NBT-SG I 溶液中的孕育时间和 NBT 的浓度。琼脂糖凝胶的染色时间比丙烯酰胺凝胶更长, 因为琼脂糖凝胶的厚度更大。这种方法在 SDS 存在时不能很好地与 NBT 沉淀物接触。如果凝胶获得紫色的背景, 我们建议准备另一种凝胶, 并减少曝光时间。如果琼脂糖凝胶是染色和没有足够的灵敏度, 建议增...

随着生物化学和分子生物学的进步, 涉及 dna 的研究需要更好的技术来分析 dna。DNA 染色的第一个方法是银染色, 这是非常敏感的, 但缺乏选择性, 不允许样品回收。后来, 荧光 dna 染色的发展使 dna 的选择性定量与样品的回收率的可能性。第一种用于 DNA 定量的荧光染料是溴溴化物1, 它是诱变性的2。然而, 现在有改进的荧光染料, 更安全, 更敏感, 如 GelRed 和 SYBR 绿色 i (SG i)3;但所有这些荧光染料都需要使用紫外线 (UV) transilluminator 或 fluorimeter。
还有其他技术可明显染色的 DNA, 如亚甲基蓝4和晶体紫5,6,7,8,9, 但所有这些都受到降低灵敏度和选择性.唑盐是有机化合物易受减少, 当这发生他们形成一个不溶性和色的甲沉淀10,11。最近, 一些二价唑盐被证明绑定到 DNA 由于其阳性唑环12。
在最近的出版物13中, 提出了一种在聚丙烯酰胺凝胶中染色和量化 DNA 的新的可见技术, 它使用了一种叫做硝基蓝唑 (NBT) 的二价唑盐和荧光染料, 如溴溴化物, GelRed,SYBR 绿我和 SYBR 金。这种反应是在蓝光或阳光的存在下进行的, 与使用紫外线 transilluminator 的 SG I 相比, 可以提高样品的回收率。本文的目的是提供一个详细的协议的染色技术使用唑盐。

<table border="0" cellpadding="0" cellspacing="0" width="785">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Nitro blue tetrazolium</td>
			<td style="width:227px;">Gold Biotechnology</td>
			<td style="width:307px;">298-83-9</td>
			<td style="width:64px;">reagent used in the staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">SYBR Green I 10000X</td>
			<td style="width:227px;">Thermo-Fisher</td>
			<td style="width:307px;">S7563</td>
			<td>reagent used in the staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Gel extraction kit</td>
			<td style="width:227px;">QIAGEN</td>
			<td style="width:307px;">28704</td>
			<td>used to extract plasmid from the gel in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">DNA ladder</td>
			<td style="width:227px;">Thermo-Fisher</td>
			<td style="width:307px;">SM 1331</td>
			<td>used to make calibration curves and as reference to cut band in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Agar Agar</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;"></td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">sodium chloride</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">1064045000</td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">yeast extract</td>
			<td style="width:227px;">Becton, Dickinson and Company</td>
			<td style="width:307px;">212750</td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">peptone</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">72161000</td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">TEMED</td>
			<td style="width:227px;">AMRESCO</td>
			<td style="width:307px;">761</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">ammonium persulfate</td>
			<td style="width:227px;">Calbiochem</td>
			<td style="width:307px;">2310</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">acrylamide</td>
			<td style="width:227px;">invitrogen</td>
			<td style="width:307px;">15512-023</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">bisacrylamide</td>
			<td style="width:227px;">SIGMA</td>
			<td style="width:307px;">146072</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Tris-base</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">1083821000</td>
			<td>used to make TAE buffer and non-denaturing polyacrylamide gel tris-HCl buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">EDTA</td>
			<td style="width:227px;">J.T. Baker</td>
			<td style="width:307px;">8993-01</td>
			<td>used to make TAE buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Acetic acid</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">1,000,632,500</td>
			<td>used to make TAE buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">agarose</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">16802</td>
			<td>used to make TAE buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">spectrophotometer</td>
			<td style="width:227px;">Tecan</td>
			<td style="width:307px;">infinite 200 pro</td>
			<td>used to quantify DNA plasmid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">water purificatiom unit</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">Elix 100</td>
			<td>use to make water type 1 (ASTM) used in spectrophotometry and DNA dilutions\</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">distilled water</td>
			<td style="width:227px;"></td>
			<td style="width:307px;">-</td>
			<td>used in gels, culture medium, stainings and general solutions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">HCl</td>
			<td style="width:227px;">J.T. Baker</td>
			<td>9535-03</td>
			<td>used to make non-denaturing polyacrylamide gel tris-HCl buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">6X DNA loading dye</td>
			<td style="width:227px;">Thermo-Fisher</td>
			<td style="width:307px;">R0610</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">5 mm Blue LED</td>
			<td style="width:227px;">Jinyuan electronics</td>
			<td style="width:307px;">SP-021</td>
			<td>light source used in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">protoboard</td>
			<td style="width:227px;">KANDH</td>
			<td style="width:307px;">KH102</td>
			<td>light source support and circuit&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">9 V battery</td>
			<td style="width:227px;">Duracell</td>
			<td style="width:307px;">-</td>
			<td>used to power the LED&#39;s</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:187px;">horizontal electrophoresis chamber</td>
			<td style="width:227px;">Biorad</td>
			<td style="width:307px;">1704486EDU</td>
			<td>used to make and run agarose gel in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">vertical electrophoresis kit</td>
			<td style="width:227px;">Biorad</td>
			<td style="width:307px;">1658002EDU</td>
			<td>used to run polyacrylamide gels in calibration curves</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">power supply</td>
			<td style="width:227px;">Biorad</td>
			<td style="width:307px;">1645050&#160;</td>
			<td>used to power the electrophoresis</td>
		</tr>
	</tbody>
</table>

dna staining method based on formazan precipitation induced by blue light exposure

DNA 染色方法是生物医学研究的重要内容。我们设计了一个简单的方法, 使 DNA 可视化到肉眼的形成一个有色沉淀。它的工作原理是浸泡丙烯酰胺或琼脂糖 DNA 凝胶在 1x (相当于2.0 µM) SYBR 绿色 i (SG i) 和0.20 毫米硝基蓝唑, 产生紫色沉淀的甲时暴露在阳光或特别蓝光。此外, 使用我们的方法染色的琼脂糖凝胶中的氨苄西林抗性质粒进行 DNA 恢复试验。用我们的方法获得的菌落数量比传统的紫外光照射法更大。所描述的方法是快速的, 特异的, 无毒的 DNA 检测, 允许生物分子的可视化, "肉眼" 没有一个 transilluminator, 是廉价和适当的现场使用。由于这些原因, 我们新的 DNA 染色方法对研究和工业都有潜在的好处。

一个紫色的甲沉淀出现在 DNA 的位置 (通过质谱13验证)。在实验中, 加载了一个 DNA 阶梯来制作一个校准曲线 (图 3)。该协议适用于丙烯酰胺和琼脂糖凝胶, 但它的强度较低, 在琼脂糖中需要较长的时间。然而, 使用琼脂糖凝胶允许回收样品使用一个商业可用的试剂盒, 它不干扰的提取。与 SG I 使用 transilluminator (图 4</stron...

Watch this Scientific Journal Video about DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure at JoVE.com

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

这种方法允许选择性染色和量化的 DNA 在凝胶中浸泡在 SYBR 绿 I/硝基蓝唑溶液, 然后暴露在阳光或蓝光光源。这会产生明显的沉淀, 几乎不需要任何设备, 这使得它非常适合于现场使用。

基于蓝光照射诱发甲沉淀的 DNA 染色方法

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Research

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解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

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Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

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

用丙烯酰胺凝胶电泳法可视化近红外荧光标记 dna 聚合酶活性

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

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Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

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Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

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

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

Analysis of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

β-淀粉脂质诱导纤维蛋白斑点结构异常的光谱分析

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

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. ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

基于原生自荧光的sFLIM测量植物部分荧光探针和木质素之间的相互作用

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...