Name: dna staining method based on formazan precipitation induced by blue light exposure
Uploaded: 2018-01-28T14:00:00.000+00:00
Duration: 8 min 11 s
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 毫升丙烯酰胺/双丙烯酰胺 (30/0.8% 瓦特/五), 0.25 毫升1米的 pH 6.8, 0.02 毫升的10% 硫酸铵, 和0.002 毫升的 TEMED。凝胶的尺寸是7.3 厘米 x 8.2 cm x 0.075 cm (l x w x d)。 注意:使用15井梳子的0.75 毫米厚度的最佳灵敏度。</li>
	</ol>
</li>
	<li>将适当数量的6x 载入缓冲液和 dna 样本添加到1.5 毫升的离心管 (例如使用1µL 的6x 加载缓冲区到5µL 的 dna 样本)。</li>
	<li>将凝胶置于垂直电泳室。<ol>
<li>用750毫升的1x 泰缓冲器填充会议厅, 以运行2凝胶。准备1升50x 泰, 使用442克的三基地, 57.1 毫升的冰醋酸, 100 毫升的0.5 米 EDTA pH 8。将蒸馏水加到最后一卷1升。</li>
		<li>将样品装入凝胶中。在100伏的2小时运行凝胶。</li>
	</ol>
</li>
	<li>或者, 用理查德·山姆布鲁克14中发现的配方制备1x 泰琼脂糖凝胶。<ol>
<li>将300毫升的1x 泰加到水平电泳室。运行凝胶45分钟, 在 100 v</li>
	</ol>
</li>
</ol>2. NBT-SG I 染色 (图 1)。
<ol><li>
丙烯酰胺凝胶染色。
	<ol>
<li>准备解决方案: 0.1% 瓦特/v NBT 和 1.2x SG i。 注意:它可以使用溴溴化物, GelRed 和 SYBR 黄金代替 sg i, 但 sg 我有最好的表现。</li>
		<li>将16.75 毫升的 1.2x SYBR 绿色 I 溶液倒入合适大小的容器中。 注意:我们使用吸管尖端盒的盖子作为容器, 并且这些容量被计算用这个容器盖胶凝体;它的尺寸是 11.6 cm x 7.6 cm x 2.6 cm (l x w x d)。</li>
		<li>将凝胶放入溶液中。添加3.25 毫升的0.1% 瓦特/v NBT 溶液给0.2 毫米 NBT 和 1x (相当于2.0 µM) SG I 最终浓度。</li>
		<li>用塑料薄膜封住容器, 用铝箔将容器完全包裹起来, 以阻挡任何环境光线。 注意:该塑料薄膜用于防止飞溅, 避免铝箔与染色液的反应。</li>
		<li>在 60 rpm 的轨道振动筛上摇动25分钟。</li>
		<li>拆卸铝塑盖。暴露在阳光下90分钟最大。检查每5分钟, 直到感兴趣的波段出现。</li>
		<li>要使用蓝光, 准备一个 protoboard 与3发光二极管 (led) 和地方的 led 约5毫米从凝胶表面 (检查染色连续, 因为需要的时间将取决于强度的 LED 光源)。</li>
	</ol>
</li>
	<li>
琼脂糖凝胶染色。
	<ol>
<li>在准备和运行1% 琼脂糖凝胶, 如1.4 节 (尺寸是1厘米 x 6.2 厘米 x 7 厘米; l x w x d), 按照相同的步骤, 在2.1 与这些修改:</li>
		<li>倒入41.9 毫升的 1.2x SG I 溶液进入吸管尖端盒的盖子;它的尺寸是 11.6 cm x 7.6 cm x 2.6 cm (l x w x d)。添加8.1 毫升的 0.1% NBT 溶液。</li>
		<li>用塑料薄膜封住容器, 然后用铝箔将容器完全包裹起来, 以阻挡任何环境光。</li>
		<li>在 60 rpm 的轨道振动筛上晃动1小时。</li>
	</ol>
</li>
</ol>3. 数据分析。
<ol><li>
NBT-SG I 校准曲线。
	<ol>
<li>通过扫描 1200 dpi 获得凝胶图像。</li>
		<li>用于图像分析的开放式软件。使用图像编辑器对图像进行伸直。运行图像密度测量。</li>
		<li>计算曲线下的面积。准备一个信号/信号Max与 DNA 浓度的关系图。</li>
	</ol>
</li>
</ol>4. DNA 恢复。
<ol><li>使用理查德·山姆布鲁克14中发现的配方, 在1x 泰秀中准备1% 琼脂糖凝胶。</li>
	<li>将300毫升的1x 泰加到水平电泳室。在图 2中给出的方案之后, 加载4µL 100 ng/µL pDJ10015质粒和 DNA 阶梯。运行1小时的凝胶 100 v</li>
	<li>马克和重量两2毫升离心管。</li>
	<li>把凝胶切成中间, 以便有两个相同的一半。每一个都应该包含阶梯和样品质粒来比较不同的染色技术。示意图表示形式在图 2中。</li>
	<li>染色一半与 NBT-SG I 如在步骤2。<ol>
<li>用干净的手术刀切出质粒带。将凝胶片保存在一根管子内并称重。</li>
	</ol>
</li>
	<li>染色的其他部分与 sg i 50 毫升的 1x sg i 溶液和缓慢晃动 (60 rpm) 在黑暗中的1小时凝胶。<ol>
<li>将凝胶放在 transilluminator 上, 并暴露2分钟, 用干净的手术刀把质粒带出来。</li>
		<li>将凝胶片保存在另一管中, 称量。 注意:该协议可以暂停在这里, 而保持在-20 ° c 的管。</li>
	</ol>
</li>
	<li>使用商业凝胶萃取试剂盒进行质粒提取。</li>
</ol>5. 质粒正常化。
<ol><li>在1.5 毫升离心管, 添加10µL 提取质粒和60µL I 型水。混合和转移到60µL 石英试管或使用2µL 直接在 Nanodrop。</li>
	<li>测量吸收光谱在230到320毫微米之间。使用以下公式计算样品的质粒浓度: <img alt="Equation 1" src="/files/ftp_upload/56528/56528eq1v3.jpg"> 其中D是应用的稀释因子 (在本例中为 70/10), Ax 是在x nm 上吸收的, 50 是用于 dsDNA 的µg/mL 的转换因子。</li>
	<li>稀释样品至 9.1 ng/µL。</li>
	<li>转换大肠杆菌(大肠杆菌) 有能力的细胞 (如XL10 金) 在井下转换过程16使用5µL 稀释样品。</li>
	<li>计算培养皿上菌落数并注意稀释。使用以下公式计算菌落形成单元 (CFU)17: <img alt="Equation 2" src="/files/ftp_upload/56528/56528eq2v2.jpg"> 其中, D是从初始区域性应用的稀释因子。</li>
	<li>执行不配对 t 检验。</li>
</ol>

<ol>
	<li>LePecq, J. B., Paoletti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescent+complex+between+ethidium+bromide+and+nucleic+acids:+physical-chemical+characterization.">A fluorescent complex between ethidium bromide and nucleic acids: physical-chemical characterization.</a> J. Mol. Biol. 27 (1), 87-106 (1967).</li><li>Singer, V. L., Lawlor, T. 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. J. 66 (A150), (1994).</li><li>Flores, N., Valle, F., Bolivar, F., Merino, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+DNA+from+agarose+gels+stained+with+methylene+blue.">Recovery of DNA from agarose gels stained with methylene blue.</a> Biotechniques. 13 (2), 203-205 (1992).</li><li>Yang, Y. I., Jung, D. W., Bai, D. G., Yoo, G. S., Choi, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counterion-dye+staining+method+for+DNA+in+agarose+gels+using+crystal+violet+and+methyl+orange.">Counterion-dye staining method for DNA in agarose gels using crystal violet and methyl orange.</a> Electrophoresis. 22 (5), 855-859 (2001).</li><li>Yang, Y. 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. D., Walter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+protein+translocation+across+the+yeast+endoplasmic+reticulum:+ATP-dependent+post-translational+translocation+of+the+prepro-&#945;-factor.">In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent post-translational translocation of the prepro-&#945;-factor.</a> Cell. 45 (3), 397-406 (1986).</li><li>Inoue, H., Nojima, H., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transformation+of+Escherichia+coli+with+plasmids.">High efficiency transformation of Escherichia coli with plasmids.</a> Gene. 96 (1), 23-28 (1990).</li><li>Madigan, M., Martinko, J., Parker, 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> Brock biology of microorganisms. , (2003).</li></ol>

作者没有什么可透露的。

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><tbody><tr><td>Nitro blue tetrazolium</td><td>Gold Biotechnology</td><td>298-83-9</td><td>reagent used in the staining</td></tr><tr><td>SYBR Green I 10000X</td><td>Thermo-Fisher</td><td>S7563</td><td>reagent used in the staining</td></tr><tr><td>Gel extraction kit</td><td>QIAGEN</td><td>28704</td><td>used to extract plasmid from the gel in integrity assay</td></tr><tr><td>DNA ladder</td><td>Thermo-Fisher</td><td>SM 1331</td><td>used to make calibration curves and as reference to cut band in integrity assay</td></tr><tr><td>Agar Agar</td><td>Merck</td><td></td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>sodium chloride</td><td>Merck</td><td>1064045000</td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>yeast extract</td><td>Becton, Dickinson and Company</td><td>212750</td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>peptone</td><td>Merck</td><td>72161000</td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>TEMED</td><td>AMRESCO</td><td>761</td><td>used to make polyacrylamide gels</td></tr><tr><td>ammonium persulfate</td><td>Calbiochem</td><td>2310</td><td>used to make polyacrylamide gels</td></tr><tr><td>acrylamide</td><td>invitrogen</td><td>15512-023</td><td>used to make polyacrylamide gels</td></tr><tr><td>bisacrylamide</td><td>SIGMA</td><td>146072</td><td>used to make polyacrylamide gels</td></tr><tr><td>Tris-base</td><td>Merck</td><td>1083821000</td><td>used to make TAE buffer and non-denaturing polyacrylamide gel tris-HCl buffer</td></tr><tr><td>EDTA</td><td>J.T. Baker</td><td>8993-01</td><td>used to make TAE buffer</td></tr><tr><td>Acetic acid</td><td>Merck</td><td>1,000,632,500</td><td>used to make TAE buffer</td></tr><tr><td>agarose</td><td>Merck</td><td>16802</td><td>used to make TAE buffer</td></tr><tr><td>spectrophotometer</td><td>Tecan</td><td>infinite 200 pro</td><td>used to quantify DNA plasmid</td></tr><tr><td>water purificatiom unit</td><td>Merck</td><td>Elix 100</td><td>use to make water type 1 (ASTM) used in spectrophotometry and DNA dilutions\</td></tr><tr><td>distilled water</td><td></td><td>-</td><td>used in gels, culture medium, stainings and general solutions</td></tr><tr><td>HCl</td><td>J.T. Baker</td><td>9535-03</td><td>used to make non-denaturing polyacrylamide gel tris-HCl buffer</td></tr><tr><td>6X DNA loading dye</td><td>Thermo-Fisher</td><td>R0610</td><td></td></tr><tr><td>5 mm Blue LED</td><td>Jinyuan electronics</td><td>SP-021</td><td>light source used in integrity assay</td></tr><tr><td>protoboard</td><td>KANDH</td><td>KH102</td><td>light source support and circuit&amp;nbsp;</td></tr><tr><td>9 V battery</td><td>Duracell</td><td>-</td><td>used to power the LED&#039;s</td></tr><tr><td>horizontal electrophoresis chamber</td><td>Biorad</td><td>1704486EDU</td><td>used to make and run agarose gel in integrity assay</td></tr><tr><td>vertical electrophoresis kit</td><td>Biorad</td><td>1658002EDU</td><td>used to run polyacrylamide gels in calibration curves</td></tr><tr><td>power supply</td><td>Biorad</td><td>1645050&amp;nbsp;</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

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

dna-staining-method-based-on-formazan-precipitation-induced-blue

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

9.4K 次观看.  Universidad de Chile. 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。

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

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.

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.

用噻唑橙代替溴代或替代染料进行 DNA 电泳

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

科研

生物化学

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

荧光显微镜方法测定细菌在协会的可行性与哺乳动物细胞

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

免疫与感染

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

转换纳米粒子介导的激酶活化远程触发细胞内信号转导的集成系统

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

生物工程

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

适合与天然化合物库的探索抗生物试验的平台

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Analysis of Lipid Droplet Content in Fission and Budding Yeasts using Automated Image Processing

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are used as models in lipid metabolic research or for industrial lipid production. Lipid droplets are highly dynamic storage bodies and their cellular content represents a convenient readout of the lipid metabolic state. Fluorescence microscopy is a method of choice for quantitative analysis of cellular lipid droplets, as it relies on widely available equipment and allows analysis of individual lipid droplets. Furthermore, microscopic image analysis can be automated, greatly increasing overall analysis throughput. Here, we describe an experimental and analytical workflow for automated detection and quantitative description of individual lipid droplets in three different model yeast species: the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus, and the budding yeast Saccharomyces cerevisiae. Lipid droplets are visualized with BODIPY 493/503, and cell-impermeable fluorescent dextran is added to the culture media to help identify cell boundaries. Cells are subjected to 3D epifluorescence microscopy in green and blue channels and the resulting z-stack images are processed automatically by a MATLAB pipeline. The procedure outputs rich quantitative data on cellular lipid droplet content and individual lipid droplet characteristics in a tabular format suitable for downstream analyses in major spreadsheet or statistical packages. We provide example analyses of lipid droplet content under various conditions that affect cellular lipid metabolism.

Here, we present a MATLAB implementation of automated detection and quantitative description of lipid droplets in fluorescence microscopy images of fission and budding yeast cells.

使用自动图像处理分析裂变和萌芽酵母中的脂滴含量

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are ...

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can be generated from FMN photolysis upon irradiation with visible light. The ROS generated from FMN photolysis are harmful to microorganisms, including pathogenic bacteria such as Staphylococcus aureus (S. aureus). This article presents a protocol for deactivating S. aureus, as an example, via photochemical reactions involving FMN under visible light irradiation. The superoxide radical anion (<img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" />) generated during the FMN photolysis is evaluated via nitro blue tetrazolium (NBT) reduction. The microbial viability of S. aureus that is attributed to reactive <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" /> species was used to determine the effectiveness of the process. The bacterial inactivation rate is proportional to FMN concentration. Violet light is more efficient in inactivating S. aureus than blue light irradiation, while the red or green light does not drive FMN photolysis. The present article demonstrates FMN photolysis as a simple and safe method for sanitary processes.

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Here, we present a protocol to inactivate pathogenic bacteria with reactive oxygen species produced during photolysis of flavin mononucleotide (FMN) under blue and violet light irradiation of low intensity. FMN photolysis is demonstrated to be a simple and safe method for sanitary processes.

通过可见光光解 核 黄素-5′-磷酸灭活病原体

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

使用荧光抗生素探针对细菌耐药性的可视化

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

活体细胞荧光显微镜观察微生物细胞生长过程中的基本过程

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

生物学

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

延时结构照明显微镜线粒体核素的特定标签

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...

基于蓝光照射诱发甲沉淀的 DNA 染色方法

摘要

探索更多视频

此视频中的章节

用噻唑橙代替溴代或替代染料进行 DNA 电泳

荧光显微镜方法测定细菌在协会的可行性与哺乳动物细胞

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

转换纳米粒子介导的激酶活化远程触发细胞内信号转导的集成系统

适合与天然化合物库的探索抗生物试验的平台

使用自动图像处理分析裂变和萌芽酵母中的脂滴含量

通过可见光光解核黄素-5′-磷酸灭活病原体

使用荧光抗生素探针对细菌耐药性的可视化

活体细胞荧光显微镜观察微生物细胞生长过程中的基本过程

延时结构照明显微镜线粒体核素的特定标签

基于蓝光照射诱发甲沉淀的 DNA 染色方法

摘要

探索更多视频

此视频中的章节

用噻唑橙代替溴代或替代染料进行 DNA 电泳

荧光显微镜方法测定细菌在协会的可行性与哺乳动物细胞

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

转换纳米粒子介导的激酶活化远程触发细胞内信号转导的集成系统

适合与天然化合物库的探索抗生物试验的平台

使用自动图像处理分析裂变和萌芽酵母中的脂滴含量

通过可见光光解 核 黄素-5′-磷酸灭活病原体

使用荧光抗生素探针对细菌耐药性的可视化

活体细胞荧光显微镜观察微生物细胞生长过程中的基本过程

延时结构照明显微镜线粒体核素的特定标签

通过可见光光解核黄素-5′-磷酸灭活病原体