基于蓝光照射诱发甲沉淀的 DNA 染色方法
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Nitro blue tetrazolium | Gold Biotechnology | 298-83-9 | reagent used in the staining |
| SYBR Green I 10000X | Thermo-Fisher | S7563 | reagent used in the staining |
| Gel extraction kit | QIAGEN | 28704 | used to extract plasmid from the gel in integrity assay |
| DNA ladder | Thermo-Fisher | SM 1331 | used to make calibration curves and as reference to cut band in integrity assay |
| Agar Agar | Merck | used to make LB-ampicillin culture plates | |
| sodium chloride | Merck | 1064045000 | used to make LB-ampicillin culture plates |
| yeast extract | Becton, Dickinson and Company | 212750 | used to make LB-ampicillin culture plates |
| peptone | Merck | 72161000 | used to make LB-ampicillin culture plates |
| TEMED | AMRESCO | 761 | used to make polyacrylamide gels |
| ammonium persulfate | Calbiochem | 2310 | used to make polyacrylamide gels |
| acrylamide | invitrogen | 15512-023 | used to make polyacrylamide gels |
| bisacrylamide | SIGMA | 146072 | used to make polyacrylamide gels |
| Tris-base | Merck | 1083821000 | used to make TAE buffer and non-denaturing polyacrylamide gel tris-HCl buffer |
| EDTA | J.T. Baker | 8993-01 | used to make TAE buffer |
| Acetic acid | Merck | 1,000,632,500 | used to make TAE buffer |
| agarose | Merck | 16802 | used to make TAE buffer |
| spectrophotometer | Tecan | infinite 200 pro | used to quantify DNA plasmid |
| water purificatiom unit | Merck | Elix 100 | use to make water type 1 (ASTM) used in spectrophotometry and DNA dilutions |
| distilled water | – | used in gels, culture medium, stainings and general solutions | |
| HCl | J.T. Baker | 9535-03 | used to make non-denaturing polyacrylamide gel tris-HCl buffer |
| 6X DNA loading dye | Thermo-Fisher | R0610 | |
| 5 mm Blue LED | Jinyuan electronics | SP-021 | light source used in integrity assay |
| protoboard | KANDH | KH102 | light source support and circuit |
| 9 V battery | Duracell | – | used to power the LED's |
| horizontal electrophoresis chamber | Biorad | 1704486EDU | used to make and run agarose gel in integrity assay |
| vertical electrophoresis kit | Biorad | 1658002EDU | used to run polyacrylamide gels in calibration curves |
| power supply | Biorad | 1645050 | used to power the electrophoresis |
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