Name: a western blotting protocol for small numbers of hematopoietic stem cells
Uploaded: 2018-08-22T12:30:00.000+00:00
Duration: 8 min 45 s
Description: Watch this Scientific Journal Video about A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells at JoVE.com

<p class="jove_content">美国国立卫生研究院资助 R01 CA149976 支持这项工作。</p>

<p class="jove_content">所有程序必须按照机构动物使用和护理指南执行。该程序是为分析小鼠造血干细胞和祖细胞 (HSCs 和高压), 但可以适应其他细胞群体的分析而制定的。</p>
<p class="jove_title">1. 流式细胞仪分离小鼠 HSCs 和高压的研究</p>
<ol><li>收获小鼠骨髓细胞如描述在文献<sup class="xref">6</sup>。<br>注: 在<strong class="xfig">图 1</strong>和<strong class="xfig">图 2</strong>所示的数据中, 骨髓是从 C57BL/6J 小鼠身上采集的。</li>
	<li>按照制造商的说明, 使用鼠标沿袭损耗套件执行骨髓细胞的血统耗尽。</li>
	<li>用抗体对细胞表面的标记进行孵化, 如<sup class="xref">7</sup>所述。在<strong class="xfig">图 1</strong>和<strong class="xfig">图 2</strong>所示的实验中, 使用了对 Sca-1、试剂盒、Flt3 和沿袭 (林) 标记 Mac1、Gr1、CD3e、B220 和 Ter119 的抗体。</li>
	<li>排序 2000-2万林<sup> </sup>Sca1<sup>+</sup>Flt3 细胞<sup></sup> (HSCs<sup></sup> ) 或林<sup>-</sup>Sca1 试剂盒<sup>+</sup>细胞<sup></sup>(hp) 1.5 毫升离心管含有0.1 毫升磷酸盐缓冲盐水 (PBS) 与2% 胎牛血清。<br>注意: 对于<strong class="xfig">图 1</strong>和<strong class="xfig">图 2</strong>所示的数据, 细胞使用了一个带有70µM 喷嘴的流式分析仪进行排序。最大采集量为1.4 毫升。</li>
</ol><p class="jove_title">2. 样品准备</p>
<p class="jove_content">注意: 此步骤非常关键。仔细处理样品。清除上清液时, 要小心不要扰乱细胞颗粒。</p>
<ol><li>离心机的1.5 毫升管包含排序的细胞在摆动斗转子在4°c, 500 x g, 5 分钟. 用吸管和100µL 吸管提示从细胞颗粒中取出所有但大约20µL。不要扰乱球团。<ol>
<li>如果样品包含少于5000个细胞, 并且被排序的样品的总容量少于0.2 毫升, 转移细胞首先到低捆绑0.2 毫升 PCR 管在离心之前。</li>
		<li>如果样品包含少于5000个细胞和容量是超过0.2 毫升, 用离心机在4°c, 500 x g, 为5分钟旋转细胞下来, 并且去除所有, 但200µL 的上清。再将剩余的200µL 中的颗粒细胞重新悬浮, 然后将细胞转移到0.2 毫升的 PCR 管中, 再次旋转。在第二次旋转后从颗粒中除去 20-30 µL, 然后重新悬浮细胞。</li>
	</ol>
</li>
	<li>并用重悬在试管中左上清的细胞。如有必要, 添加额外的 PBS 在2% 的血清/pbs (你也可以使用2% 牛血清白蛋白 (BSA)/pbs, 或 pbs 单独) 获得浓度 5 x10<sup>4</sup>至 5<sup></sup> x 10 细胞/毫升。<br>注: 使用细胞计数法收集, 以估计用于悬浮细胞的体积。</li>
	<li>使用 2-5 µL 的重新悬浮细胞, 以确定准确的细胞浓度使用自动单元计数器 (按照制造商的指示) 或 hemocytometer<sup class="xref">9</sup>。</li>
	<li>将含有所需细胞数的重悬浮细胞转移到新的1.5 毫升管中。在<strong class="xfig">图 1</strong>, 2000, 1000, 或 500 HSCs 或高压的实验被转移了。所需的细胞数取决于由西方印迹获得的信号的强度, 并根据经验确定。使用20µL 或100µL 离心吸管提示进行转移。</li>
	<li>离心机在摆动斗转子中的细胞在4°c, 500 x g, 5 分钟。</li>
	<li>根据制造商的指示, 生成100x 库存解决方案, 在亚砜中溶解蛋白酶体和磷酸酶抑制剂。</li>
	<li>通过稀释制造商提供的相当于蒸馏水的 4x Laemmli 样品缓冲器, 准备 2x Laemmli 样品缓冲器。添加适量的100x 股蛋白酶体和磷酸酶抑制剂, 以获得最终浓度2x 抑制剂在 2x Laemmli 样本缓冲区。<br>注: 2x Laemmli 样本缓冲区可以提前进行, 但蛋白酶体和磷酸酶抑制剂应立即添加, 然后添加 2x Laemmli 样本缓冲区 (加上抑制剂) 的细胞颗粒溶解细胞, 如以下步骤所述。</li>
	<li>小心地从细胞颗粒中取出部分清液, 并将其与试管中剩余的上清液结合在一起, 添加等量的 2x Laemmli 样品缓冲器加上蛋白酶体和磷酸酶抑制剂以达到500的最终浓度。单元格/µL 在 1x Laemmli 示例缓冲区中。<br>注意: 例如, 如果您在步骤2.5 中将2000个单元的总体积20µL 转移到一个管, 则在离心单元格 (步骤 2.6) 后, 应从颗粒中移除上清的18µL, 并将剩余的上清的2个µL 添加相等容积 (2 µL) 2x Laemmli 样品缓冲器, 以达到500细胞/µL 在 1x Laemmli 样品缓冲器中的最终浓度。</li>
	<li>并用重悬颗粒以尽快生成裂解液。在这一点上, 样品可以立即电泳通过 SDS 页凝胶, 或可在液体 N<sub>2</sub>冻结, 并存储在-80 °c, 以供将来使用。</li>
</ol><p class="jove_title">3. 电泳</p>
<ol><li>将裂解物在95摄氏度或沸水中加热5分钟。</li>
	<li>Electrophorese 1-40 µL 的裂解物 (包含从500高达2万细胞当量) 通过 SDS 页凝胶在100V 使用标准协议<sup class="xref">10</sup>。<br>注: 在凝胶中装入的裂解液体积是由产生理想信号所需的细胞数决定的, 并取决于感兴趣的蛋白质的丰度和抗体的质量。<strong class="xfig">图 1</strong>中的数据获得了2000、1000和500个细胞当量的裂解物。井的宽度应该在3毫米到1.5 毫米的范围内. 1.5 毫米的牙齿可以用剪刀从商业可用的梳子上剪下来。</li>
</ol><p class="jove_title">4. 转移和阻止</p>
<p class="jove_content">注: 执行<sup class="xref">11</sup>标准协议后的西方污点。下面简要介绍了这些步骤:</p>
<ol><li>预处理氟 (PVDF) 膜与甲醇十年代, 然后简要地洗涤膜与蒸馏水。</li>
	<li>按照转移器具制造商提供的说明书中的说明, 将蛋白质从 SDS 页凝胶转移到膜上。</li>
	<li>转移后, 2 毫升5% 牛血清白蛋白 (BSA) 在 PBST (PBS 与0.1% 补体) 一夜间在4摄氏度以下标准协议<sup class="xref">11</sup>。</li>
</ol><p class="jove_title">5. 抗体标记</p>
<ol><li>使用供应商推荐的抗体稀释, 在4摄氏度的情况下, 用抗体在一夜床上孵育细胞膜。<br>注: 在材料表中, 我们对 HSCs 和高压的抗体以及相应的抗体稀释进行了验证。使用标准程序执行抗体标记<sup class="xref">8</sup>。</li>
</ol><p class="jove_title">6. 检测</p>
<ol><li>清洗膜3次, 每 PBST 5 分钟室温下清洗。</li>
	<li>室温下用2毫升增强化学发光缓冲剂孵化膜1分钟。根据制造商的说明或显影胶片和胶片处理器, 使用成像系统检测信号。</li>
</ol>

<ol>
	<li>Krutzik, P. O., Clutter, M. R., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinate+analysis+of+murine+immune+cell+surface+markers+and+intracellular+phosphoproteins+by+flow+cytometry.">Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry.</a> <em style='font-style: italic'>J Immunol</em>. <b>175</b> (4), 2357-2365 (2005).</li><li>Du, 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=Signaling+profiling+at+the+single-cell+level+identifies+a+distinct+signaling+signature+in+murine+hematopoietic+stem+cells.">Signaling profiling at the single-cell level identifies a distinct signaling signature in murine hematopoietic stem cells.</a> <em style='font-style: italic'>Stem Cells</em>. <b>30</b> (7), 1447-1454 (2012).</li><li>Signer, R. A., Magee, J. A., Salic, A., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematopoietic+stem+cells+require+a+highly+regulated+protein+synthesis+rate.">Haematopoietic stem cells require a highly regulated protein synthesis rate.</a> <em style='font-style: italic'>Nature</em>. <b>509</b> (7498), 49-54 (2014).</li><li>Wang, 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=A+differentiation+checkpoint+limits+hematopoietic+stem+cell+self-renewal+in+response+to+DNA+damage.">A differentiation checkpoint limits hematopoietic stem cell self-renewal in response to DNA damage.</a> <em style='font-style: italic'>Cell</em>. <b>148</b> (5), 1001-1014 (2012).</li><li>Hoshii, 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=mTORC1+is+essential+for+leukemia+propagation+but+not+stem+cell+self-renewal.">mTORC1 is essential for leukemia propagation but not stem cell self-renewal.</a> <em style='font-style: italic'>J Clin Invest</em>. <b>122</b> (6), 2114-2129 (2012).</li><li>Signer, R. A., 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=The+rate+of+protein+synthesis+in+hematopoietic+stem+cells+is+limited+partly+by+4E-BPs.">The rate of protein synthesis in hematopoietic stem cells is limited partly by 4E-BPs.</a> <em style='font-style: italic'>Genes Dev</em>. <b>30</b> (15), 1698-1703 (2016).</li><li>Cai, X., 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=Runx1+deficiency+decreases+ribosome+biogenesis+and+confers+stress+resistance+to+hematopoietic+stem+and+progenitor+cells.">Runx1 deficiency decreases ribosome biogenesis and confers stress resistance to hematopoietic stem and progenitor cells.</a> <em style='font-style: italic'>Cell Stem Cell</em>. , (2015).</li><li>Loven, 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=Revisiting+global+gene+expression+analysis.">Revisiting global gene expression analysis.</a> <em style='font-style: italic'>Cell</em>. <b>151</b> (3), 476-482 (2012).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+Hemoacytometer+to+Count+Cells.">Using a Hemoacytometer to Count Cells.</a> <em style='font-style: italic'>Basic Methods in Cellular and Molecular Biology</em>. , (2017).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separating+Protein+with+SDS-PAGE.">Separating Protein with SDS-PAGE.</a> <em style='font-style: italic'>Basic Methods in Cellular and Molecular Biology</em>. , (2017).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Western+Blot.">The Western Blot.</a> <em style='font-style: italic'>Basic Methods in Cellular and Molecular Biology</em>. , (2017).</li><li>Jackson, R. J., Hellen, C. U., Pestova, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+eukaryotic+translation+initiation+and+principles+of+its+regulation.">The mechanism of eukaryotic translation initiation and principles of its regulation.</a> <em style='font-style: italic'>Nat Rev Mol Cell Biol</em>. <b>11</b> (2), 113-127 (2010).</li><li>Eaton, S. L., 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+guide+to+modern+quantitative+fluorescent+western+blotting+with+troubleshooting+strategies.">A guide to modern quantitative fluorescent western blotting with troubleshooting strategies.</a> <em style='font-style: italic'>J Vis Exp</em>.  (93), e52099 (2014).</li></ol>

<p class="jove_content">作者没有利益冲突要申报。</p>

<p class="jove_content">西方印迹是一种常见的技术检测特定的蛋白质和激活信号通路的组织或细胞。通过对通常使用的程序进行小的调整, 我们能够例行地检测15种不同的蛋白质 (<strong>材料表</strong>), 在 1.5万 HSCs, 在某些情况下, 在 500 HSCs。该协议中最关键的步骤是: 1) 精确计数单元格, 2) 最小化管之间的传输数, 3) 株溶藻单元格直接与 Laemmli 样本缓冲区。当株溶藻的细胞颗粒与 Laemmli 样品缓冲, 我们发现, 而不是删除所有...

<p class="jove_content">造血干细胞 (HSCs) 是自我更新的细胞, 可以引起所有的血血统。它们是骨髓中相对稀少的细胞, 使得生物化学分析变得困难。适合于分析稀有细胞的方法, 如流式细胞术, 对于定量测定细胞表面标记物和胞内蛋白的相对数量非常有用。然而, 细胞内蛋白的分析需要使用细胞通透程序来实现抗体的存取, 而不是所有细胞表面的表位都能存活这些程序<sup class="xref">1</sup><sup>,</sup><sup class="xref">2</sup>。此外, 在不同的蛋白质亚型或裂解产物之间鉴别的抗体通常不能用于流式细胞术, 因此调查人员仍然依靠西方的印迹进行某些类型的分析。</p>
<p class="jove_content">裂解物细胞的印迹分析是大多数实验室的常规程序。细胞可以在保存细胞表面分子表位的本地条件下纯化, 细胞裂解物随后可以被制备和分析。然而, 用西方印迹对稀有的原代细胞种群中的蛋白质进行分析, 可以要求 euthanizing 大量的动物获得足够的细胞。通过对几个步骤进行小的调整, 传统的西方印迹协议能够检测出相对较小数量的 HSCs (500-1.5万, 取决于感兴趣的蛋白质) 的蛋白质。这些调整包括准确计数细胞, 仔细处理细胞颗粒, 减少细胞间的转移, 以最小化细胞损失, 并株溶藻一个定义数量的细胞与集中加载缓冲包含蛋白酶体和磷酸酶抑制剂。许多发表的报告包括西部污点获得与2万或更多 HSCs<sup class="xref">3</sup><sup>,</sup><sup class="xref">4</sup><sup>,</sup><sup class="xref">5</sup><sup>,</sup><sup class="xref">6</sup><sup>,</sup><sup class="xref">7</sup>;这个简单的程序将减少在4到40倍之间产生等效数据所需的细胞和实验动物的数量。该协议旨在以每个单元为基础对结果进行规范化, 而不是内部控制。这使得检测蛋白质水平的全面降低, 如果数据被规范化到内部控制, 可以忽略。本文描述了在细胞基础上规范化的重要性, 用于分析基因表达数据<sup class="xref">8</sup>, 同样的原理也适用于用西方印迹来定量蛋白质。此优化的协议对于需要分析少量单元格的任何人都是有用的。</p>

<table><tbody><tr><td>sodium dodecyl sulfate (SDS)</td><td>Fisher Scientific</td><td>BP166-500</td><td>SDS-PAGE</td></tr><tr><td>TEMED</td><td>Fisher Scientific</td><td>BP150-20</td><td>SDS-PAGE</td></tr><tr><td>30% Acrylamidel Bis solution</td><td>Bio-Rad</td><td>1610158</td><td>SDS-PAGE</td></tr><tr><td>1.5M Tris-HCl pH8.8</td><td>TEKNOVA</td><td>T1588</td><td>SDS-PAGE</td></tr><tr><td>1.0M Tris-HCl pH6.8</td><td>TEKNOVA</td><td>T1068</td><td>SDS-PAGE</td></tr><tr><td>Ammonium Persulfate</td><td>Fisher Scientific</td><td>BP179-25</td><td>SDS-PAGE</td></tr><tr><td>mini-protean 3 comb</td><td>Bio-Rad</td><td>1653360</td><td>SDS-PAGE</td></tr><tr><td>Mini-PROTEAN Tetra Cell</td><td>Bio-Rad</td><td>1658005EDU</td><td>SDS-PAGE</td></tr><tr><td>Transfer System</td><td>Bio-Rad</td><td>1704155EDU</td><td>transfer</td></tr><tr><td>PowerPac HC Power Supply</td><td>Bio-Rad</td><td>1645052EDU</td><td>electrophoresis</td></tr><tr><td>Imaging System</td><td>Bio-Rad</td><td>1708195</td><td>signal detection</td></tr><tr><td>4x Laemmli Sample Buffer</td><td>Bio-Rad</td><td>1610747EDU</td><td>sample preparation</td></tr><tr><td>10x Tris/Glycine/SDS Electrophoresis Buffer</td><td>Bio-Rad</td><td>1610732EDU</td><td>electrophoresis</td></tr><tr><td>2-Mercaptoethanol</td><td>Bio-Rad</td><td>1610710EDU</td><td>sample preparation</td></tr><tr><td>RTA Mini PVDF Transfer Kit, for 40 blots</td><td>Bio-Rad</td><td>1704272</td><td>transfer</td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Sigma</td><td>11697498001</td><td>sample preparation</td></tr><tr><td>Phosphatase Inhibitor Cocktailrs</td><td>Gold Biotechnology</td><td>GB-450-1</td><td>sample preparation</td></tr><tr><td>EIF4G</td><td>Cell signaling</td><td>2469</td><td>WB antibody, validated in 1000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): Fig2</td></tr><tr><td>p-Rps6</td><td>Cell signaling</td><td>4858</td><td>WB antibody,validated in 1000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): Fig2</td></tr><tr><td>actin(HRP conjugated)</td><td>abcam</td><td>ab49900</td><td>WB antibody,validated in 500 cells&lt;br/&gt; antibody concentration: 1:5000&lt;br/&gt; reference(figure): Fig1, Fig2</td></tr><tr><td>LC-3</td><td>Abcam</td><td>ab48394</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig5)</td></tr><tr><td>S6</td><td>Cell Signaling</td><td>2317</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>4EBP1</td><td>Cell Signaling</td><td>9644</td><td>WB antibody, validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>p-4EBP1</td><td>Cell Signaling</td><td>2855</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>TSC2</td><td>Cell Signaling</td><td>4308</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>HSP90</td><td>Cell Signaling</td><td>4877</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig5)</td></tr><tr><td>PTEN</td><td>Cell Signaling</td><td>9188</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>mTOR</td><td>Cell Signaling</td><td>2983</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>p-mTOR</td><td>Cell Signaling</td><td>5536</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>PP2AB</td><td>Cell Signaling</td><td>4953</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>p-AMPKa</td><td>Cell Signaling</td><td>2535</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>actin</td><td>Santa Cruz</td><td>sc-47778</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:3000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>lineage depletion kit (mouse)</td><td>Miltenyi Biotec</td><td>130-090-858</td><td>hematopoietic stem and progenitor cells purification</td></tr><tr><td>LS columns</td><td>Miltenyi Biotec</td><td>130-041-305</td><td>hematopoietic stem and progenitor cells purification</td></tr><tr><td>SCF</td><td>PeproTech</td><td>250-03</td><td>phosphorylation stimulation</td></tr><tr><td>APC-Cy7 c-kit antibody</td><td>ThermoFisher</td><td>A15423</td><td>cell sorting</td></tr><tr><td>anti-B220</td><td>ThermoFisher</td><td>48-0452-82</td><td>cell sorting</td></tr><tr><td>anti-CD3e</td><td>ThermoFisher</td><td>48-0031-82</td><td>cell sorting</td></tr><tr><td>anti-Mac1</td><td>ThermoFisher</td><td>48-0112-82</td><td>cell sorting</td></tr><tr><td>anti-Gr1</td><td>ThermoFisher</td><td>48-5931-82</td><td>cell sorting</td></tr><tr><td>anti-Ter119</td><td>ThermoFisher</td><td>48-5921-82</td><td>cell sorting</td></tr><tr><td>Spacer Plates with 1.0 mm Integrated Spacers</td><td>Bio-Rad</td><td>1653311</td><td>SDS-PAGE</td></tr><tr><td>BSA</td><td>Research Products International</td><td>A30075</td><td>membrane blocking</td></tr><tr><td>serum</td><td>Atlanta Biologicals</td><td>A12450</td><td>medium supplement</td></tr><tr><td>cell counter</td><td>Invitrogen</td><td>AMQAF1000</td><td>cell counting</td></tr><tr><td>hemocytometer</td><td>ThermoFisher</td><td>&amp;nbsp;S17040</td><td>cell counting</td></tr><tr><td>flim</td><td>Denville Scientific</td><td>E3012</td><td>signal detection</td></tr><tr><td>medical film processor</td><td>Konica</td><td>SRC-101A</td><td>signal detection</td></tr><tr><td>Supersignal West Femto Maximum Sensitivity Substrate</td><td>Therom Scientific</td><td>34096</td><td>signal detection</td></tr><tr><td>Allegra X-15R Centrifuge (Rotor SX4750A)</td><td>Beckman Coulter</td><td>X-15R</td><td>sample preparation</td></tr><tr><td>BLUEstain protein ladder</td><td>Gold Biotechnology</td><td>P007-500</td><td>electrophoresis</td></tr><tr><td>cell sorter</td><td>BD</td><td>FACSAria II</td><td>sample preparation</td></tr><tr><td>Incublock</td><td>Denville Scientific</td><td>I0510</td><td>electrophoresis</td></tr></tbody></table>

a western blotting protocol for small numbers of hematopoietic stem cells

<p class="jove_content">造血干细胞 (HSCs) 是罕见的细胞, 与小鼠骨髓中仅含有2.5万表型的长期再生 HSCs。对 HSCs (500-1.5万细胞) 的小数目进行了优化, 并适合于对其进行分析。表型 HSCs 纯化, 准确计数, 直接裂解 Laemmli 样品缓冲。用月桂酸钠聚丙烯酰胺凝胶电泳 (SDS 页) 分析了含有等量细胞的裂解物, 并根据标准的西方印迹协议制备并处理了该印迹。使用此协议, 可以对 2000-5000 HSCs 进行例行分析, 在某些情况下, 数据可以从很少500个单元中获得, 而大多数出版物中报告的2万到4万个单元格。本议定书应普遍适用于其他造血细胞, 并能通过标准实验室程序对少量细胞进行常规分析。</p>

<p class="jove_content" fo:keep-together.within-page="1">500-2000 纯化 HSCs 和高压钠灯的代表性结果如<strong class="xfig">图 1</strong>和<strong class="xfig">图 2</strong>所示。<strong class="xfig">图 1</strong>中的β肌动蛋白信号可以从从一只老鼠的骨髓中纯化出来的 500 HSCs 和高压钠灯中检测出来。请注意, 将裂解物装入1.5 毫米井, 产生了比加载到3.0 毫米井更强的500高压信号。<strong class="xfig">图 2</strong>是 EIF4G 的西方印迹和 Rps6 (p-Rps6) 的磷酸化, ?...

Watch this Scientific Journal Video about A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells at JoVE.com

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

<p class="jove_content">通过对500只造血干细胞或祖细胞进行分析, 优化了标准的西方印迹协议。优化包括仔细处理细胞样本, 限制管道之间的传输, 并直接株溶藻 Laemmli 样本缓冲区中的细胞。</p>

一种用于小数目造血干细胞的西方印迹协议

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

a-western-blotting-protocol-for-small-numbers-hematopoietic-stem

Research

JoVE Journal

Biochemistry

10.3K 次观看.  Cincinnati Children's Hospital Medical Center. 通过对500只造血干细胞或祖细胞进行分析, 优化了标准的西方印迹协议。优化包括仔细处理细胞样本, 限制管道之间的传输, 并直接株溶藻 Laemmli 样本缓冲区中的细胞。

视频: A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Buffers

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of Solutions<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">To set up for this lab experiment, wear the appropriate personal protective equipment, including a lab coat, chemical splash goggles, and gloves. All solutions for this lab should be prepared in a chemical fume hood.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare 100 mL of a 1 M NaOH solution. <strong>Note: </strong>NaOH is strongly corrosive, so use caution while handling it.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Measure 4 g of solid NaOH and pour it in a 125-mL Erlenmeyer flask. Then, add 50 mL of deionized water to the flask. Stir the mixture on a stir plate to dissolve the NaOH.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Label a 100-mL polyethylene bottle as &#39;1 M NaOH&#39;. Once the NaOH has dissolved and the solution appears homogeneous, add another 50 mL of deionized water. Then, retrieve the stir bar and transfer the solution to the labeled bottle. Place the capped bottle in the back of the instructor&#39;s hood.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Prepare 10 mL of a 0.56 mM solution of neutral red in deionized water. Measure 1.6172 mg of neutral red into a 10-mL volumetric flask.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Use a pipette to fill the flask about 2/3 full with deionized water. Cover the flask with plastic paraffin film and agitate the mixture until the neutral red dissolves. Then, remove the film and fill the flask to the marked line. Place a micro-stir bar in the flask and stir the solution until it appears homogeneous.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Label a 20-mL vial as &#39;0.56 mM neutral red&#39; and transfer the solution to the vial. Cap the vial and store it in the fridge.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Prepare 10 mL of a 0.66 mM solution of riboflavin-binding protein in the same way, starting by weighing 198 mg of the protein. Transfer the solution to a labeled 20-mL vial and store the solution in the fridge.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Prepare or obtain a set of buffers. Here, we will go through the preparation of 100 mL of 50 mM pH 6.5 monosodium phosphate buffer, adjusted with 1 M NaOH as an example.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Measure 0.5995 g of anhydrous monosodium phosphate into a 125-mL Erlenmeyer flask. Add 80 mL of deionized water to the flask and begin stirring the mixture.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">While the mixture stirs, turn on and calibrate a digital pH meter.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Once the salt has dissolved and the solution appears homogeneous, clamp the pH sensor in the buffer and wait for the reading to stabilize.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Confirm that the 1 M NaOH solution has cooled to room temperature. Then, slowly add NaOH to the stirring buffer solution with pauses to let the pH stabilize until the buffer reaches pH 6.5. This may only take 1 or 2 mL of NaOH. Then, turn off the stir motor, rinse the pH probe, and retrieve the stir bar.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Pour the pH adjusted buffer into a clean 100-mL volumetric flask and bring the solution up to the 100-mL mark with deionized water. Seal the flask with plastic paraffin film and invert it until the solution appears homogeneous.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Label a clean 125-mL polyethylene bottle as &#39;50 mM monosodium phosphate buffer pH 6.5&#39;. Transfer the solution to the bottle and cap it. Store the buffer in the instructor&#39;s hood.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Prepare additional buffers in a similar fashion, using 1 M NaOH or 2 M HCl as appropriate to adjust the pH. Alter the buffer concentrations as needed to achieve roughly the same ionic strength.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">When finished, store the 1 M NaOH in a corrosives cabinet, clean the glassware and equipment using your standard procedures, and throw out disposable items in the lab trash.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Preparation of the Laboratory<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Bring a container of anhydrous monosodium phosphate to the analytical balance area and confirm that there are sufficient supplies of weighing paper, laboratory wipes, and clean spatulas.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Set out the following lab equipment and glassware at each lab station (we suggest that students work in pairs):
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Stir plate</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Lab stand with thermometer clamp</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;pH meter</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Handheld spectrophotometer</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Calibration buffers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;400-mL beakers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;100-mL beakers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;25-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;50-mL graduated cylinder</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;10-mL graduated cylinders</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Small watch glass</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Magnetic stir bar</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Funnel</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Forceps</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Plastic wash bottle</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;250- or 500-mL capped polyethylene bottle</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Roll of laboratory tape</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Plastic pipettes</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Labeling pen</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Box of laboratory wipes</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;1-mL micropipette with tips</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;200-&#181;L micropipette with tips</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;10&#160;&#160;&#160;&#160;1.5-mL cuvettes with caps</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Set out additional disposable plastic pipettes so that everyone will have easy access to them.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Place labeled bottles or flasks containing the buffers in a central area.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Label a 1-mL micropipette for each buffer and place the micropipettes with the corresponding buffers along with a box of pipette tips.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Portion out the neutral red and protein solutions. Label three 5-mL test tubes as &#39;neutral red&#39; and three 5-mL test tubes as &#39;riboflavin-binding protein&#39;. Then, take the vials of neutral red solution and riboflavin-binding protein from the fridge and place about 2 mL of the neutral red solution and 2 mL of riboflavin-binding protein solution in the appropriately labeled test tubes.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Set out the neutral red and protein solutions so the student groups can share the test tubes.</div>
		</li>
	</ul>
	</li>
</ol>

Buffers: Common Ion Effect, Henderson-Hasselbalch Equation, and Buffer Capacity - Prep

缓冲区

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Preparation of SolutionsExpand
	Here, we show the laboratory preparation for 10 students ...

教育

JoVE Lab Manual

化学

Lab Manual Sub Articles

Thin-Layer Chromatography

<p>Source: Lara Al Hariri and&#160;Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Effects of Mobile Phase Composition on Compound Separation<button class="expand">Expand</button>
	<p>In this section of the lab, you will use TLC to compare how well pure hexane and a mixture of 60% hexane and 40% ethyl acetate can separate phenol, benzoic acid, and butyl phenyl ether by measuring the distance the compound traveled in each solvent.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Before starting the lab, put on a lab coat, safety glasses, and nitrile gloves. Note: Hexane and ethyl acetate are both volatile, toxic, and flammable, so always work with them in a fume hood. Change your gloves if you get solvent on them, as hexane and ethyl acetate will permeate nitrile gloves within minutes.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Now, obtain a paper towel and lay it on the benchtop. Carefully pick up a TLC plate using tweezers at the very end of the plate.<strong> Note: </strong>Always hold TLC plates by the edges or at the very top with tweezers to avoid damaging or contaminating the silica gel.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Look for chips or scratches in the silica gel and get a new plate if necessary. It&#39;s fine if there is a little chipping at only one end, since that can be the top.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Place the plate on the paper towel in your fume hood, and lay a metric ruler next to it. Use a graphite pencil to gently make a small mark on the silica gel ~1 cm from the top of the plate. Write &#8216;100&#8217; on the plate above the mark.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Next, gently mark the plate 1 cm above the bottom on both sides. Carefully trace a straight line between the bottom marks to make the starting line.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Then, add 3 evenly spaced small marks to the starting line with the outer marks at least 0.5 cm from the sides of the plate. <strong>Note:</strong> It&#39;s important for the silica gel to be undamaged here, so if you scratch it with the pencil, prepare a new plate.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Now, trace the outline of the TLC plate in your lab notebook and precisely copy the marks on the plate. Label the three marks as &#8216;phenol&#8217;, &#8216;benzoic acid&#8217;, and &#8216;butyl phenyl ether&#8217;. You may also want to label the marks on the plate itself.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Prepare a second TLC plate labeled &#8216;60/40&#8217; in the same way. Now, bring the labeled plates to the common bench and locate the 1 mg/mL solutions of phenol, benzoic acid, and butyl phenyl ether in ethanol.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Dip a clean capillary into one of the three solutions. Briefly and lightly dab the capillary squarely against the intersection between the starting line and the mark for that compound. You should spot twice for each compound.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Use the same capillary to spot the same solution on your second TLC plate. The spots should be no more than 2 mm in diameter. Then, discard the capillary and close the vial.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Spot the other two solutions on the plates, in the same way, using clean capillaries. Be careful not to cross-contaminate the solutions. <strong>Note</strong>: If a spot is very light or very small, wait for it to dry, and then spot the same solution on top of the dry spot. If the spot is still wet, capillary action could make it too wide and dilute.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Once you have made three spots on each plate, discard the capillaries and bring the plates back to your hood. Keep the plates lying flat while they dry.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Now, label a 50-mL jar &#8216;100% hexane&#8217; and bring the jar, its cap, and a 10-mL graduated cylinder to the solvent hood.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Measure 2 &#8211; 3 mL of pure hexane with your graduated cylinder and pour it into the jar to fill it to a depth of 0.62 &#8211; 0.7 cm. Cap the bottle and jar and return to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Label a second 50-mL jar &#8216;60% hexane, 40% ethyl acetate&#8217; and bring it and a clean graduated cylinder to the solvent hood. Measure 2 &#8211; 3 mL of the 60/40 mixture into the jar and return to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Stand the plates next to the jars to compare the relative position of the solvent and the spots. The solvent level should be below the spots for each plate.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Then, obtain three pieces of filter paper, put a filter paper upright against the wall in each jar, and then cap the jars tightly.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Initial each plate above the mark so you can identify them later. Confirm that the filter papers in the jars have become saturated with solvent and that the spots on the plates are completely dry.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Now, open the 100% hexane jar and use tweezers to gently place the 100 plate in the jar, keeping the plate level with the solvent surface as it enters the solvent. Close the jar tightly, being careful not to jostle the plate.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Leave the jar still as the solvent travels up the plate. Development in pure hexane usually takes about 7 &#8211; 10 min. Keep an eye on the plate to make sure that the solvent doesn&#39;t reach the top.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">When the pure hexane solvent front is about 1 cm from the top of the plate, retrieve it using tweezers. Lay it on the paper towel with the silica gel facing up and quickly mark the solvent front with a line and pencil across the plate. Leave the plate lying flat as the solvent evaporates.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Place the other plate in the 60/40 eluent in the same way. Wait until the solvent front of the 60/40 plate is about 1 cm from the top, and then retrieve it and quickly mark the solvent front in the same way.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Once the 100% hexane plate is completely dry, pick it up with tweezers and bring it and a pencil to the UV lamp on the common bench.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Place the plate under the lamp and illuminate it with UV light. The plate will glow green because of a fluorescent marker in the silica gel layer. The dark spots are the compounds.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Carefully outline every spot in pencil, even if they haven&#39;t moved very far.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Then, turn off the lamp and bring your plate to the iodine development hood. Open the iodine chamber. Use tweezers to gently place the plate upright in the iodine chamber, and then cover the chamber again.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Wait 3 &#8211; 4 min for the iodine vapor to stay in the spots. Then, retrieve the plate with tweezers, close the chamber, and outline the brown and yellow spots on the plate. These spots will fade, so mark them quickly.</div>
		</li>
		<li data-sub-note-item="1-29">
		<div class="lab-step">Visualize the dry 60/40 plate under UV light and then with iodine vapor in the same way.<strong> Note: </strong>To avoid side reactions, always check plates under UV light before exposing them to iodine.</div>
		</li>
		<li data-sub-note-item="1-30">
		<div class="lab-step">Now, precisely copy the spots and the solvent fronts onto the corresponding drawings of the plates in your lab notebook. For each plate, measure and record the height of the solvent front relative to the starting line.</div>
		</li>
		<li data-sub-note-item="1-31">
		<div class="lab-step">Lastly, measure the distance between the center of each spot and its starting line, and record it in your lab notebook. You can now move on to the next part of the lab.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Unknown Compound Identification<button class="expand">Expand</button>
	<p>In the second half of the lab, you&#39;ll use TLC to find out what&#39;s in an unknown analgesic. It could contain one or more of these compounds: aspirin, caffeine, and acetaminophen. These compounds are highly polar, so you&#39;ll use a premade mixture of 90% ethyl acetate and 10% hexane as the eluent.</p>
	<p>You&#39;ll run the three reference compounds and the unknown all on the same plate. Later, you&#39;ll use the Rf values of the reference compounds to identify the unknown. First, double-check which unknown material you were assigned to analyze before starting.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-3">
		<div class="lab-step">Now, lay a clean paper towel in the hood and obtain a new TLC plate. Prepare the top mark and the starting line the same way as before. Make four evenly spaced ticks on the plate, making sure that the outer marks are at least 0.5 cm away from the edges.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Draw an exact copy of the plate in your lab notebook and label the tick marks as &#8216;aspirin&#8217;, &#8216;acetaminophen&#8217;, &#8216;caffeine&#8217;, and &#8216;unknown&#8217;.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Now, bring your plate to the common bench. Spot solutions of aspirin, acetaminophen, caffeine, and the unknown that you were assigned on the appropriate marks using a clean capillary for each solution. <strong>Note: </strong>When you are spotting the unknown solution, don&#39;t dip the capillary too far into the solution vial to avoid clogging your capillary with insoluble material from the unknown substance.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Discard the capillaries and close the vials before bringing the plate back to your fume hood. Place the plate on a paper towel to finish drying.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Next, label a clean 50-mL screw-top jar &#8216;90% ethyl acetate, 10% hexane&#8217;, and bring it in a clean graduated cylinder to the solvent hood.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Place 2 &#8211; 3 mL of the 90/10 solution in the jar and return to your fume hood. Confirm that the solvent level will be below the spots on the plate. Then, place a clean filter paper upright in the jar and close it tightly.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Once the TLC plate is dry and the filter paper is saturated, carefully place the plate in the solvent. <strong>Note:</strong> It&#39;s essential to hold the plate level when you do this. If the solvent front is diagonal, the solvent distance traveled will be different for each spot, making it impossible to visually compare the unknown to the reference spots.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Wait for the solvent front to reach 1 cm from the top of the plate. Then, retrieve the plate, mark it solvent front, and let it dry while lying flat.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Once the plate is dry, visualize it with UV light and in the iodine chamber, and outline the spots in pencil.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">The unknown material could be a mixture of compounds, so look carefully for spots in line with all three reference spots.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Then, precisely copy the spots onto the drawing of the plate in your lab notebook. Measure and record the height of the solvent front and the spots relative to the starting line.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">When you are finished, retrieve the solvent-saturated filter papers from the jars and let them dry in the hood.</div>
		</li>
		<li data-sub-note-item="2-15">
		<div class="lab-step">Pour leftover solvents into the standard organic waste, wash your glassware following your lab&#39;s usual methods, and put away your lab equipment.</div>
		</li>
		<li data-sub-note-item="2-16">
		<div class="lab-step">Throw out used TLC plates in the lab trash or with glass waste, depending on the backing material.</div>
		</li>
		<li data-sub-note-item="2-17">
		<div class="lab-step">Lastly, once the filters are dry, throw them out along with any remaining trash.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="3-1">
		<div class="lab-step">Calculate the R<sub>f</sub> values for phenol, benzoic acid, and butyl phenyl ether eluted with 100% hexane. For each compound on the TLC plate, measure the distance from the starting line to the center of its spot. Then, divide this value by the distance from the starting line to the solvent front.</div>
		</li>
		<li data-sub-note-item="3-2">
		<div class="lab-step">Only butyl phenyl ether moved a measurable distance from the starting line, which is consistent with it being the least polar. For effective separation, the R<sub>f</sub> values should differ by 0.3 &#8211; 0.7. Thus, we can infer that phenol and benzoic acid are too polar to be separated effectively by an apolar solvent like pure hexane.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Calculate the R<sub>f</sub> values for the plate that you ran in a 60/40 mixture of hexane and ethyl acetate. Ethyl acetate is a moderately polar solvent, so the compounds traveled farther and are spaced farther apart on the plate.</div>
		</li>
		<li data-sub-note-item="3-4">
		<div class="lab-step">The separation order shows that benzoic acid, the most polar compound, had the highest affinity for silica gel, whereas butyl phenyl ether, the least polar compound, had the lowest affinity for silica gel. This is consistent with the assumption that less polar compounds travel farther on polar stationary phases.</div>
		</li>
		<li data-sub-note-item="3-5">
		<div class="lab-step">Look at your third plate and calculate the R<sub>f</sub> values for aspirin, acetaminophen, and caffeine. Caffeine, the most polar complex of the three, traveled the least. Acetaminophen traveled the second farthest, while aspirin, the least polar, traveled the most.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Finally, identify the compounds in your unknown material by comparing its spot or spots to the three reference spots.</div>
		</li>
	</ul>
	</li>
</ol>

Thin-Layer Chromatography: Experimental Setup and Separation - Procedure

薄层色谱

Source: Lara Al Hariri and&#160;Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

	Effects of Mobile Phase Composition on Compound ...

Concentration Dependence

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Concentration Dependence<button class="expand">Expand</button>
	<p>In this experiment, you will combine varying concentrations of aqueous HCl and sodium thiosulfate to make solid sulfur, which rapidly gathers into opaque yellow particles at a certain sulfur concentration. Since the reaction solution starts clear and colorless, you can easily tell when it reaches that concentration.</p>
	<p>You&#39;ll use the same test tube and total volume each time, so it will take the same amount of sulfur to make each solution completely opaque. Thus, you&#39;ll measure the reaction progress by timing how long that takes. You&#39;ll then use that data to estimate the reaction orders of the individual reactants and of the overall reaction.</p>
	<p>Before starting the lab, make a table listing the reactant concentrations, the time to solution opacity, and the solution temperature for the trials.</p>
	<h4><strong>Table 1: Reaction progress</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td><strong>Trial</strong></td>
				<td><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] (M)</strong></td>
				<td><strong>[HCl] (M)</strong></td>
				<td><strong>Time (s)</strong></td>
				<td><strong>Temp (&#176;C)</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">1</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">2</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">3</td>
				<td style="text-align: center;">0.2 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">4</td>
				<td style="text-align: center;">0.15 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">5</td>
				<td style="text-align: center;">0.05 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">6</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">6.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">7</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">4.5 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">8</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">1.5 M</td>
				<td></td>
				<td></td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/50/Table_1._Reaction_progress.xlsx" target="_blank">Click Here to download Table 1</a></p>
	<p>We want to make sure that the reactions are all taking place at room temperature. You&#39;ll perform two benchmark trials, three trials with different sodium thiosulfate concentrations, and three trials with different HCl concentrations. You will vary the concentrations by diluting stock solutions of sodium thiosulfate and HCl, as shown in the following tables.</p>
	<h4><strong>Table 2: Sodium thiosulfate dilutions</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td style="text-align: center;"><strong>Target Concentration</strong></td>
				<td style="text-align: center;"><strong>Volume of 0.2 M sodium thiosulfate</strong></td>
				<td style="text-align: center;"><strong>Volume of DI water</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">0.05 M</td>
				<td style="text-align: center;">5 mL</td>
				<td style="text-align: center;">15 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.10 M</td>
				<td style="text-align: center;">10 mL</td>
				<td style="text-align: center;">10 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.15 M</td>
				<td style="text-align: center;">15 mL</td>
				<td style="text-align: center;">5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.20 M</td>
				<td style="text-align: center;">20 mL</td>
				<td style="text-align: center;">0 mL</td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/50/Table_2._Sodium_thiosulfate_dilutions.xlsx" target="_blank">Click Here to download Table 2</a></p>
	<h4><strong>Table 3: HCl Dilutions</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td style="text-align: center;"><strong>Target Concentration</strong></td>
				<td style="text-align: center;"><strong>Volume of 6.0 M HCl</strong></td>
				<td style="text-align: center;"><strong>Volume of DI water</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">1.5 M</td>
				<td style="text-align: center;">2.5 mL</td>
				<td style="text-align: center;">7.5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">3.0 M</td>
				<td style="text-align: center;">5.0 mL</td>
				<td style="text-align: center;">5.0 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">4.5 M</td>
				<td style="text-align: center;">7.5 mL</td>
				<td style="text-align: center;">2.5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">6.0 M</td>
				<td style="text-align: center;">10 mL</td>
				<td style="text-align: center;">0 mL</td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/50/Table_3._HCl_Dilutions.xlsx" target="_blank">Click Here to download Table 3</a></p>
	<p>Remember to use caution when handling HCl, which is toxic and highly corrosive. Sulfur dioxide, which is a gaseous product of this reaction, is also toxic. You will leave the reaction waste in the fume hood overnight to let the sulfur dioxide escape harmlessly.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-6">
		<div class="lab-step">First, put on a lab coat, splash-proof safety glasses, and nitrile gloves.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Fill a plastic wash bottle with deionized water and get a test tube marked with an X.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Clamp the test tube over the center of a small stir plate. The label must be on the opposite side from you. Make sure that you can clearly see the X. Place a small magnetic stir bar in the test tube.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Use a thermometer clamp to secure a glass thermometer in the lower third of the tube, keeping it clear of the X and the stir bar.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Cut a sheet of about 20 squares of plastic paraffin film from a roll and bring the sheet back to your hood.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Lay paper towels in the fume hood as a clean surface for labware to be reused.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Label a 600-mL beaker as &#8216;waste&#8217; for the reaction, a 100-mL beaker as &#8216;0.2 M sodium thiosulfate&#8217;, and a 50-mL beaker as &#8216;6 M HCl&#8217;.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Fill the 100-mL beaker with 0.2 M sodium thiosulfate from the stock bottle in the dispensing hood.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">After that, bring the 50-mL beaker and a watch glass to the acid-dispensing hood to obtain 50 mL of 6 M HCl. Cover the filled beaker with the watch glass and bring your portion of HCl back to your hood.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Prepare 20 mL of 0.1 M sodium thiosulfate for the first benchmark trial. Use a 10-mL volumetric pipette to measure 10 mL of 0.2 M sodium thiosulfate and dispense it into a 20-mL volumetric flask. Fill the 20-mL volumetric flask with deionized water to the mark.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Then, cut a square of plastic paraffin film, seal the flask, and invert it several times to thoroughly mix the solution.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Remove the film and pour the solution into the test tube.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Prepare 10 mL of 3 M HCl. Pipette 5 mL of 6 M HCl into a 10-mL graduated cylinder. Add deionized water to fill the graduated cylinder to the 10-mL line.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Seal the top of the graduated cylinder with plastic paraffin film and thoroughly mix the HCl solution by inverting it several times.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Now, start the stir motor and get a reset stopwatch. Confirm that you can see the X through the solution in the test tube.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Simultaneously start the stopwatch and pour the 3 M HCl into the test tube.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Watch the X through the solution, which will start becoming cloudy when enough sulfur has been produced to saturate it. Stop the stopwatch exactly when the solution becomes so opaque that the X disappears.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Record the time in your lab notebook for benchmark trial 1 and then reset the stopwatch. Also, record the temperature of the solution.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Now, turn off the stir motor, remove the thermometer, and rinse it with deionized water into the waste beaker.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Then, empty the test tube into the waste beaker, retrieve the stir bar with long forceps, and rinse the test tube and stir bar with deionized water.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Thoroughly clean the test tube with a test tube brush. Briefly rinse the volumetric flask and graduated cylinder with deionized water as well. Dry everything with paper towels.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Repeat the trial in the exact same way for the second benchmark time. If the time is not within 3 - 5 s of the first trial, repeat the benchmark trial until you have two consistent trials.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Follow the same overall procedure for each subsequent trial, changing the dilutions as indicated in your table and cleaning your glassware between each trial. Use a 5-mL volumetric pipette as needed when measuring the sodium thiosulfate stock solution in trials four and five.</div>
		</li>
		<li data-sub-note-item="1-29">
		<div class="lab-step">Once you have performed all eight trials, rinse your glassware and tools one last time and neutralize the reaction waste with baking soda. Use caution because sulfur dioxide is one of the reaction products.</div>
		</li>
		<li data-sub-note-item="1-30">
		<div class="lab-step">Transfer the neutralized reaction waste to the designated waste container. Then, pour any remaining sodium thiosulfate solution into its labeled waste container and rinse the beaker with deionized water.</div>
		</li>
		<li data-sub-note-item="1-31">
		<div class="lab-step">Neutralize any remaining HCl with baking soda before flushing it down the drain with tap water.</div>
		</li>
		<li data-sub-note-item="1-32">
		<div class="lab-step">Finally, clean and put away your labware according to your standard practices and throw out any trash remaining in your hood. Your instructor will dispose of the reaction waste later.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Now, estimate the reaction order of sodium thiosulfate and HCl. Calculate the starting concentration of sodium thiosulfate in the reaction mixture for trials 1 through 5. Looking at the concentration of sodium thiosulfate and the reaction times, we see that as the concentration doubles, the time to opacity is halved.
		<h4><strong>Table 4: Estimating reaction order sodium thiosulfate</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Trial</strong></td>
					<td style="text-align: center;"><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] added</strong></td>
					<td style="text-align: center;"><strong>Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub> volume (mL)</strong></td>
					<td style="text-align: center;"><strong>Total volume (mL)</strong></td>
					<td style="text-align: center;"><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] in mixture </strong></td>
					<td style="text-align: center;"><strong>Time (s)</strong></td>
					<td style="text-align: center;"><strong>Inverse time (s<sup>-1</sup>)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1, 2</td>
					<td style="text-align: center;">0.1</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">3</td>
					<td style="text-align: center;">0.2</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">4</td>
					<td style="text-align: center;">0.15</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">5</td>
					<td style="text-align: center;">0.05</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/50/Table_4._Estimating_reaction_order_sodium_thiosulfate.xlsx" target="_blank">Click Here to download Table 4</a></div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Remember that each trial used the same volume, so it took the same amount of sulfur to make each solution opaque. Thus, the reaction is twice as fast when the sodium thiosulfate concentration is doubled.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">When the concentration increases by a factor of 4, the reaction rate also increases by a factor of 4. This one-to-one relationship suggests that the reaction was first order for sodium thiosulfate. If so, we would expect a plot of rate versus sodium thiosulfate concentration to be linear.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">We didn&#39;t measure how much sulfur it took to turn the solution opaque, but we assume that it was the same for each trial. This lets us use the inverse of time to opacity as a stand-in for the rate. The slope won&#39;t be equal to k, but that&#39;s OK for this lab.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Plot inverse reaction time as a function of sodium thiosulfate concentration. This confirms the linear relationship between sulfur production and thiosulfate concentration. Thus, the reaction seems to be first order with respect to thiosulfate.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Use the same method to estimate the reaction order of HCl. First, calculate the starting concentration of HCl in the benchmark trials and in trials 6 through 8.
		<h4><strong>Table 5: Estimating reaction order HCl</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Trial</strong></td>
					<td style="text-align: center;"><strong>[HCl] added</strong></td>
					<td style="text-align: center;"><strong>HCl volume (mL) </strong></td>
					<td style="text-align: center;"><strong>Total volume (mL) </strong></td>
					<td style="text-align: center;"><strong>[HCl] in mixture </strong></td>
					<td style="text-align: center;"><strong>Time (s)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1, 2</td>
					<td style="text-align: center;">0.1</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">3</td>
					<td style="text-align: center;">0.2</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">4</td>
					<td style="text-align: center;">0.15</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">5</td>
					<td style="text-align: center;">0.05</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/50/Table_5._Estimating_reaction_order_HCl.xlsx" target="_blank">Click Here to download Table 5</a></div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Then, look at the concentration of HCl and the reaction times for these trials. The reaction times are nearly identical, so the HCl concentration has no effect on the reaction rate. This means that HCl seems to be zeroth order for this reaction.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Lastly, add up the reaction orders of the reactants to get the overall reaction order.</div>
		</li>
	</ul>
	</li>
</ol>

Concentration Dependence and Reaction Order - Procedure

浓度依赖性

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Concentration DependenceExpand
	In this experiment, you will combine varying concentrations of ...

Galvanic Cells

Galvanic Cells: Construction and Reduction Potential Measurement - Procedure

原电池

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Constructing a Lead-Copper Galvanic CellExpand
	In this experiment, you will construct a ...

Electrolytic Cells

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Electrolytic Cells<button class="expand">Expand</button>
	<p>In this experiment, you will be assembling an electrolytic cell to perform electroplating. An electrolytic cell contains an anode and cathode connected by a power source, which drives the nonspontaneous redox reaction in the cell. When the anode material is oxidized, the ions travel to the cathode, where they are reduced and plated.</p>
	<p>In the case of this experiment, the electrolytic cell contains a copper anode and a brass key for the cathode. Oxidation of the anode creates copper(2+) ions, which are then reduced on the brass key, forming a thin plating of solid copper.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Put on gloves, safety goggles, and a lab apron, which must be worn at all times.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Obtain one 10-ohm resistor, one copper sheet electrode, one copper wire, and one piece of emery paper.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Use the emery paper to polish the brass key and the copper sheet. Rinse the key and the copper electrode with water. The brass key will be the cathode, and the copper sheet will be the anode.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Take one 50-mL beaker and a watch glass to the instructor&#39;s hood and obtain approximately 20 mL of acetone. Place the watch glass on top of the beaker to prevent evaporation and bring it back to your bench.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Label the other 50-mL beaker as &#39;organic waste&#39;.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Use a disposable pipette to rinse the copper electrode and key with acetone. Collect excess in the waste beaker. Place the key and copper electrode on a paper towel to dry.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Measure the initial masses of the key and copper electrode and record their values in your lab notebook.
		<h4><strong>Table 1: Moles of Cu Transferred</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td></td>
					<td style="text-align: center;"><strong>Mass of key (g) </strong></td>
					<td style="text-align: center;"><strong>Change in mass (g) </strong></td>
					<td style="text-align: center;"><strong>Moles Cu plated </strong></td>
					<td style="text-align: center;"><strong>Mass of Cu electrode (g) </strong></td>
					<td style="text-align: center;"><strong>Change in mass (g) </strong></td>
					<td style="text-align: center;"><strong>Moles Cu lost </strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">0 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">5 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">1<sup>st</sup> 30 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">2<sup>nd</sup> 30 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/53/Table_1._Moles_of_Cu_Transferred.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Set up the electrolytic cell. Use one electrical wire with alligator clips to connect the negative terminal on the battery to the current probe. Wrap the 10-ohm resistor around the other end of the current probe.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Use another wire to connect the positive terminal on the battery to the copper electrode.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Wrap the copper wire around the brass key and use an electrical wire to connect the resistor on the current probe to the copper wire on the brass key.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Obtain about 200 mL of concentrated copper sulfate in a 250-mL beaker. The amount does not need to be exact. Bring the beaker back to your bench and set it on the stir plate. Carefully, place a magnetic stir bar in the beaker and turn the stir setting on to high.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Connect the data acquisition system to the current probe and allow the data acquisition system to sense the probe. It should return a reading in amps. Change the rate to two scans per min and the duration to 5 min.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Place the brass key in the beaker. Hang it off the side of the beaker so that the key is almost completely submerged but does not touch the magnetic stir bar. <strong>Note: </strong>The copper wire should not be in the solution.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Place the copper electrode in the beaker, bending it so that it hangs over the edge of the beaker. Ensure that it is not touching the stir bar or the brass key.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Start the scan and let it collect data for 5 min. This will act as a test run to make sure plating occurs on the brass key.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">After the scan ends, remove the key and copper electrode. The copper plating on the brass key should be evident. If it is not visible, check your circuit to make sure that the battery is connected in the correct orientation.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Once you have completed a successful 5-min plating, disconnect the key and copper electrode from the wiring. Remove the copper wire from the key and rinse the key and copper electrode with water and then acetone. Lay them on a paper towel to dry.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Measure the masses of the key and the copper electrode and record them in your lab notebook.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Reconnect the key and copper electrode and place them back in the solution like before. Restart the run on the data acquisition system, making sure that the rate is set to two scans per min. With the duration set to 30 min, allow the electroplating process to run.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">After the scan is complete, remove the electrode and key from the solution, disconnect them from the wires, and rinse them with water and then acetone. Allow them to dry on a paper towel, then weigh them and record their masses in your notebook.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Reassemble the setup and repeat the electroplating for a second 30-min trial. When the scan is complete, remove the key and copper electrode, rinse them like before, and record their mass.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Save the recorded data as a text file on a flash drive.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">To clean up, turn the stir setting off and remove the stir bar from the solution using forceps. Dispose of the acetone in the organic waste container provided by your instructor. The copper sulfate solution can be reused for future labs, so pour the concentrated copper sulfate back into the carboy.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Rinse all of your glassware with water and return it to the instructor.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">First, calculate the total charge Q that flowed through the system during the different trials. One coulomb is the quantity of electricity flowing through a wire carrying 1 ampere of current in 1 s, thus Q = I x t.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Review the current probe data for the first 5-min electroplating trial.<strong> Note: </strong>If the current dropped to 0 or close to 0 at the end of the trial, then the electrons stopped flowing from the copper electrode to the key. Only use the values recorded before the drop in current occurred. If the current drop to 0 early in the plating process, repeat the trial.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">The current varies over time, so calculate the average current, I.
		<h4><strong>Table 2: Moles of Electrons Transferred</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Trial</strong></td>
					<td style="text-align: center;"><strong>Time (s) </strong></td>
					<td style="text-align: center;"><strong>Current<sub>average</sub> (A) </strong></td>
					<td style="text-align: center;"><strong>Total charge (C) </strong></td>
					<td style="text-align: center;"><strong>Moles (e?) </strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">5 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">1<sup>st</sup> 30 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">2<sup>nd</sup> 30 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">Total</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/53/Table_2._Moles_of_Electrons_Transferred.xlsx" target="_blank">Click Here to download Table 2</a></div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Use the average current over the total time to determine the total charge transferred during electroplating, Q. About 20 coulombs were transferred during the 5-min trial, and between 110 and 120 coulombs were transferred during each 30-min trial. The total charge transferred over the entire 65-min electroplating process was about 250 coulombs.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Use the total charge transferred to calculate the moles of electrons plated during each trial. The moles of electrons plated equals the total charge transferred divided by the Faraday constant. The Faraday constant represents the magnitude of electric charge per mole of electrons and is determined by multiplying the charge of an electron by Avogadro&#39;s constant.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Determine the moles of electrons that were transferred for the 5-min and 30-min trials.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Calculate the moles of copper that were plated on the key after each trial. Similarly, determine the moles of copper lost from the copper sheet electrode.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Compare the moles of copper that were lost from the electrode to the amount that was plated to the brass key. The numbers should be close.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Check to see if these values match what is expected based on the number of moles of electrons that were transferred. The reduction of 1 mole of copper(2+) ions requires 2 moles of electrons to produce 1 mole of solid copper.</div>
		</li>
	</ul>
	</li>
</ol>

Electrolytic Cells: Construction and Electroplating - Procedure

电解槽

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Electrolytic CellsExpand
	In this experiment, you will be assembling an electrolytic cell to ...

Extraction

Extraction: Liquid-Liquid and Acid-Base Extraction - Procedure

萃取

Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

	Cellulose RecoveryExpand
	Extraction and filtration can ...

Cell Division

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Observing the Cell Cycle in a Root Tip
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Hypotheses: The experimental hypothesis is that in root tips slices that have been treated with nocodazole, a chemical that interferes with microtubular polymerization, all of the cells will be arrested at the same stage of the cell cycle and that in untreated onion tip slices all of the different stages of the cell cycle will be visualized. The null hypothesis is that there will be no differences in mitosis between the two groups and that different stages of the cell cycle will be visualized. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To prepare for this experiment you will use an onion that has been placed in water for several days until new white root tips have begun to sprout. Use a razor blade to cut a 1 cm piece from the end of one of the root tips. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Place the slice of onion into a microcentrifuge tube labeled &#8220;N&#8221; for the nocodazole condition. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then, cut a second 1 cm piece of onion root tip and place in a microcentrifuge tube labeled &#8220;C&#8221; for the control condition.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Add 2 &#181;L of the 5 mg/mL nocodazole to 1 mL of water in the microcentrifuge tube labeled N and 1 mL of water only to the tube labeled C. 
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Leave the tubes containing the root tips to incubate for 12 hours and wipe down the workspace with 70% ethanol.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">When incubation is complete, set a heat block to 60 &#186;C.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Wash the nocodazole treated tips with water and then aspirate the liquid.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Repeat this wash two more times, for a total of three washes. 
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Fill both tubes with 1 mL of one normal hydrochloric acid. Incubate the tubes in a 60 &#186;C heat block for 12 - 15 min, then discard the acid into a waste flask.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Now, add 1 mL of distilled water drop wise to the tubes at least three times to rinse the samples.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Now add one microliter of DAPI stain to 10 mL of phosphate buffered saline (PBS).
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Then place 50 &#181;L of this dilute DAPI stain into each tube, and leave them at room temperature to incubate for 12 min. 
</div></li><li data-sub-note-item="1-14"><div class="lab-step">After this, preform three distilled water washes as in step 10. 
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Label one microscope slide as 'Control' and another as 'Nocodazole'. 
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Transfer the stained roots onto their respective microscope slides and add a drop of water to the control slide and a drop of nocodazole to the nocodazole slide.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Use the razor blade to remove the unstained portions of each root tip and place a glass cover slip over each sample.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Carefully press down on each cover slip with a gloved fingertip, and then let the slides sit for 10 min. 
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Finally, view the slides under the 40X objective of a fluorescence microscope and capture images of your root tip cell preparations.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Understanding the Loss of Cell Cycle Control
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">NOTE: Before cells can pass through all of the stages of the cell cycle there are several checkpoints they must go through. These checkpoints are regulated by cyclin and cyclin-dependent kinase proteins, or CDKs. If the cyclins and CDKs determine that previous cellular events have not been adequately completed they will arrest the cells at this step and prevent them from proliferating. In cancer cells one or several of these molecular restraints for regulating cell division are lost and this allows the damaged cells to escape from cell proliferation control. These abnormal cells develop into tumors which are masses of uncontrolled proliferating cells that interfere with the normal functions of the body. Notice how before it divides this cancer cell rounds up and refracts light very strongly under the light microscope. This is because not all cancer cells successfully divide into daughter cells after each round of mitosis. This will mean that they often contain an excess of organelles and DNA and this increase in cellular content means they will refract light more intensely than normal cells do. 
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Examine your collected images and record in Table 1 the different cell structures and stages that could be identified in the onion tip slice that was treated with nocodazole. <a href="https://www.jove.com/CDNSource/lm/labs/10/Table_1_Cell_division.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="3-2"><div class="lab-step">If there are any cells undergoing mitosis, note what stages of cell division you see.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Record the percentage of cells in each stage of mitosis in Table 1. 
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Next, examine the images of the untreated onion tip slice, and record what cell structures can you identify. 
</div></li><li data-sub-note-item="3-5"><div class="lab-step">If there are any cells undergoing mitosis, consider what stages of cell division you see, and record the percentages of cells in each stage of mitosis in the Table 1. 
</div></li></ul></li></ol>

Cell Division: Observing Cell Cycle in a Root Tip - Procedure

细胞分裂

Observing the Cell Cycle in a Root Tip
ExpandHypotheses: The experimental hypothesis is that in root tips slices that have been treated with nocodazole, a ...

生物学

细胞结构与功能

Cell Structure

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Visualizing Onion and Human Cells
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">In this experiment you will prepare different types of cells from a plant and animal, onion and human respectively, and then visualize them under a microscope. HYPOTHESES: The experimental hypothesis is that the cells will appear different in overall shape and membrane structure, but there will be some shared similar structures, like nuclei, in both cells. The null hypothesis of the experiment is that there will be no difference in structure between the two cell types.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">First, to prepare an animal cell slide, start by pouring 30 mL of distilled water into a beaker.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, use a glass dropper to place 2 - 3 drops of the water onto the center of a clean microscope slide.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Next, take a clean toothpick and scrape the inside of your cheek to gather cells.  CAUTION: Do not scrape your cheeks too aggressively. The scraping should not cause discomfort or bleeding. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Put the end of the toothpick with the cheek cells onto the wetted area of the slide and mix the water and cheek cells together.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Next, add two drops of methylene blue staining solution to the wetted area of the slide and mix it in with a clean toothpick.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Let the mixture sit at room temperature for 3 minutes.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">After 3 minutes have passed, use a glass dropper to add a drop of glycerol to the mixture.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Carefully, place a cover slip over the mix on the slide by first standing one end of the cover slip vertically and aligning it along to one side of the mixture. Then, carefully lower the other end of the cover slip down until the mixture is completely covered.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Finally, remove any excess liquid with blotting paper.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Next, prepare a slide of plant cells by placing five drops of distilled water into a clean watch glass.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, with forceps, take a thin strip of onion and place it into the water.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Apply 5 drops of safranin solution to another watch glass.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Now using a pair of forceps transfer the piece of onion from the distilled water into the safranin.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Allow the onion to soak for 30 seconds.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, again with forceps, move the onion back into the distilled water.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Now, place 3 drops of glycerol onto the center of a glass microscope slide.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Using the forceps again, transfer the onion into the glycerol on the slide.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Gently place a cover slip over the onion on the slide and blot away any excess liquid with a piece of blotting paper.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Next, you will visualize the slides you have prepared using a compound microscope. First, turn on the scope and set the objective lens to the lowest level of magnification.  NOTE: A compound microscope typically has two levels of magnification.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Place the cheek tissue slide onto the stage of the microscope and bring the cells into focus by adjusting the stage and using the coarse adjustment knob.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">When the cells are in focus, observe the individual cells and draw what you see. Repeat these observations at medium and high magnifications adjusting the focus with the fine adjustment knob. IMPORTANT: Do NOT use the coarse adjustment knob at high magnifications. 
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Next, rotate the nose piece so that the oil lens is almost in place.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Place a drop of immersion oil on top of the cover slip of the cheek cell slide.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Use the oil lens to view the cheek cells and record your observations.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Repeat these steps to view the onion plant cell, moving again from low to medium, high, and oil lenses (steps 20 &#8211; 24).
</div></li><li data-sub-note-item="1-27"><div class="lab-step">Record all of your observations and note the differences you see between animal and plant cells.
</div></li><li data-sub-note-item="1-28"><div class="lab-step">When you are finished, place the cheek cell slide and toothpicks into a diluted bleach solution. Dispose of the cover slips into a glass waste container.
</div></li><li data-sub-note-item="1-29"><div class="lab-step">Finally, turn off the microscope and clean the lenses with lens cleaning paper.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Calculate the total magnification for each of the viewing levels. Remember to account for both the eyepiece magnification and the objective lens magnification. NOTE: Eye piece: 10X magnification, Objective lens: 4X, 10X, and 40X magnification
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Then, identify the structural differences between plant and animal cells using the observations made under the microscope.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Label the nuclei in both cell types and then highlight or note any differences that you see between them.
</div></li></ul></li></ol>

Cell Structure: Visualizing Onion and Human Cells - Procedure

单元结构

Visualizing Onion and Human Cells
ExpandIn this experiment you will prepare different types of cells from a plant and animal, onion and human ...

科学探究

Microbial and Fungal Diversity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Examining Microbial Colony Structures
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To begin, collect your allocated culture plates and then remove the Parafilm and lid from one of them.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Place the uncovered plate under a dissecting microscope and then observe the individual communities within each quadrant of the plate.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">As the colonies are identified, fill out the observed colony properties in the appropriate column in the table.  <a href="https://www.jove.com/CDNSource/lm/labs/28/Table_1_Microbial_diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then, repeat the observations for each of the other two bacterial species.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">To perform Gram staining of your three bacterial species, first label the ends of three microscope slides with the initials of each bacteria species, your initials, and the date. HYPOTHESES: In this experiment, the experimental hypothesis might be that it is possible to elucidate different bacterial species and shapes using Gram staining, and that Gram-positive bacteria will stain purple with crystal violet whereas Gram-negative bacteria will appear pink due to the safranin counterstain. The null hypothesis could be that all of the bacterial types will retain both stains equally, and staining will not help elucidate their species or shape.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">To begin the first stain, attach a clip to the slide for ease of handling. For each bacterial smear, remember to be sure to use a cleaned and flame-sterilized inoculating loop.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Place two loopfuls of sterile distilled water onto the center of the slide.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">After sterilizing the loop again, cool the loop by touching an uninoculated portion of the agar several times.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, remove a small amount of culture from an isolated colony and mix it with the water on the slide.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Allow the slide to dry at room temperature and heat-fix the smear by quickly passing it through a flame.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Once dry, pipette several drops of crystal violet onto the slide and let it sit for two to three minutes.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Carefully rinse the slide with distilled water by aiming the direct flow towards the top of the slide, allowing the water to gently flow down. Do not aim the water flow directly at the smear.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Next, cover the slide with Gram&#39;s iodine, leave it to sit again for two minutes, and then gently rinse with distilled water.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Decolorize the slide with 95% ethanol until stain no longer washes away.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Immediately, rinse the slide with distilled water. This will limit over-decolorizing.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, add several drops of safranin counterstain and leave for 30 s. This will stain any Gram-negative cells present.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Gently rinse the slide with distilled water and blot dry with absorbent paper.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">After staining the other two slides in the same manner (steps 6 &#8211; 17), observe each with a microscope.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Use a low power objective first to make coarse adjustments. When you find an ideal section, move on to the higher magnifications.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">After further imaging and adjusting the smear with a medium power objective, add immersion oil directly to the smear. NOTE: The oil is needed for high power objectives that will provide the best micrographs.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Now, use colored pencils to draw the observed bacteria in the appropriate magnification circles and fill in the observed characteristics in the appropriate column, noting the bacteria&#39;s shape and whether it is Gram-positive, identified by darker purple staining, or Gram-negative, identified with pink staining. <a href="https://www.jove.com/CDNSource/lm/labs/28/Table_2_Microbial_diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Finally, repeat the observation, drawing, and characterization for the other two stained bacterial slides.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Examining Fungal Structures
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Remove the <em>S. cerevisiae</em> plate from the incubator and place a drop of water onto a microscope slide.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Using a sterilized inoculating loop, smear a film of yeast into the drop of water and place a cover slip over the slide.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Place the slide under the microscope at the lowest magnification, increasing the magnification to the oil immersion setting to see the individual yeast cells.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Observe the general shape and structure of the yeast and identify any cells that are undergoing asexual reproduction by budding.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Now, draw the cells in the appropriate magnification circles.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">When you are done observing the yeast, replace the <em>S. cerevisiae</em> slide with a prepared Ascomycetes apothecium slide and set the microscope to the lowest magnification.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Using colored pencils, draw the structures at low and high magnifications, including the asci and ascospores.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Finally, place a button mushroom, <em>Agaricus bisporus</em>, under the dissecting microscope and draw the features, including the pileus, or cap, the gills, and the stipe, or stalk.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Using the coloring of the bacteria recorded in the table, determine which species are Gram-positive and which are Gram-negative.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Then, look at the shapes recorded in the table to determine which bacterial species are bacillus, coccus, or spirillum.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Next compare your illustrations of the yeast cells to your illustrations of the bacterial cells. NOTE: Yeast from the Ascomycetes phylum, such as <em>S. cerevisiae</em>, can reproduce asexually, which can be observed through the budding of the cells under a microscope.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Record any observations of yeast cells budding.  NOTE: Ascomycetes can also reproduce sexually via mitosis and haploid spore formation.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Record any observations of spore or asci formation you observed in the table.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">NOTE: Fungus from the Basidiomycota phylum exhibit clearly observable gills under the pileus.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Record any fungal species that demonstrated gills under the pileus.
</div></li></ul></li></ol>

Microbial Colony and Fungal Diversity - Procedure

微生物和真菌多样性

Examining Microbial Colony Structures
ExpandTo begin, collect your allocated culture plates and then remove the Parafilm and lid from one of them.
Place ...

进化

DNA Isolation and Restriction Enzyme Analysis

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">DNA Isolation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In this experiment you will perform DNA isolation under two experimental conditions: one using a buffer containing the detergent SDS and one without detergent. Hypotheses: The alternate hypothesis for this experiment might be that the sample prepared without SDS, a strong anionic detergent that breaks apart cell membranes, will yield less DNA than the sample prepared with SDS. The null hypothesis for this experiment might be that both samples will yield an equal amount of DNA.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To begin collect a tissue sample for DNA isolation. Here you will be using your own cheek cells. Use a Popsicle stick to scrape the insides of your cheeks for approximately 30 s. IMPORTANT: Do not scrape hard enough to cause pain or draw blood.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Position a funnel in a 15-mL conical tube. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then take 10 mL of the 1% saline solution provided to you in a labeled cup and swish it around in your mouth without swallowing. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Continue swishing for 90 s and then spit the saline solution containing your cheek cells into the funnel to collect in the conical tube.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Next, label two empty microcentrifuge tubes with your initials. 
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Label one of the tubes as +SDS and the other as -SDS to differentiate the two treatments. 
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Then add 1.5 to 2 mL of the saline solution containing the cheek cells to each tube.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Spin the tubes in a microcentrifuge for 30 s. 
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Remove the tubes from the microcentrifuge and carefully pour off the supernatant without disturbing the cells collected at the bottom of each tube. 
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Fill the tubes with more of the saline solution containing the cheek cells and repeat the centrifugation. 
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Repeat this process taking care to evenly distribute cells between the +SDS and -SDS tubes until all of the cheek cells have been collected.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Next, add 1 mL of lysis buffer containing SDS to the tube marked as +SDS, and then resuspend the cell pellet by pipetting up and down. 
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Then add 1 mL of lysis buffer without SDS to the tube marked with -SDS and resuspend the cell pellet by pipetting up and down.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">After this add 10 &#181;L of proteinase K to each tube and then carefully place the tubes in the 56 &#186;C water bath for 10 min. 
</div></li><li data-sub-note-item="1-16"><div class="lab-step">After 10 minutes carefully remove the tubes from the water bath and add 100 &#181;L of 2.5 M NaCl to each tube. 
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Close the tubes firmly and invert several times to mix.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Next, label one 15 mL conical tube as +SDS and another with -SDS. 
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Pour the contents of each microcentrifuge tube into the corresponding 15 mL conical tube. 
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Obtain one 10 mL aliquot of 100% ethanol from the freezer. 
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Hold one of the conical tubes at an angle and slowly dispense 1 mL of 100% ethanol into the tube to form a layer on top of the liquid already in the tube.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Repeat this process for the second conical tube. 
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Now look at both of your tubes. There should be a stringy precipitate present at the interface of the ethanol and the aqueous layer below it. This stringy material contains the isolated DNA. Take pictures of the DNA in the tube and make clear descriptive notes and drawings of your observations in your notebook. 
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Using a glass stir rod, pick up the DNA from the +SDS tube and examine it. Repeat this for the -SDS tube, again recording your observations. Take note of whether one of the tubes appears to have more precipitate than the other, and why this might be.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Finally, discard all liquid waste into the appropriate waste container and return all tools to their proper locations in the lab.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Restriction Enzyme Virtual Digest
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To study how restriction enzymes work, first navigate to the New England Biolabs website: <a href="https://www.neb.com/" target="__blank">https://www.neb.com/</a>
</div></li><li data-sub-note-item="2-2"><div class="lab-step">The resources available at this site allow researchers to run virtual experiments before they are performed in the lab. Click on &#8220;Tools and Resources&#8221; to find the NEBcutter tool and open the program.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">To the right of the search box find the list that says &#8220;Viral + phage&#8221;. From that list select Lambda. This is the name of the DNA you will be digesting. Select the Linear option where the tool says &#8220;the sequence is&#8221; and the &#8220;NEB enzymes&#8221; option where the tool says, &#8220;Enzymes to use&#8221;. Then click Submit.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">The next page will display a full restriction map of the lambda DNA. This restriction map can be used to predict what will happen if certain enzymes are used. Next, click on &#8220;Custom digest&#8221; under the &#8220;Main options&#8221; heading.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Search for the enzyme StuI and note that it works optimally in buffer 2.1 or CS buffer. Select the StuI enzyme and click digest.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">In the next window click on &#8220;View gel&#8221; under the &#8220;Main options&#8221; header. Select the option to run a virtual 1% gel using a pBR322-BstN1 digest.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">After the results appear, take a screenshot and paste it into a blank document.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">After this, navigate back to the main page of the NEBcutter tool and select &#8220;New custom digest&#8221;.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">This time search for the BsrGI enzyme and note that it performs optimally in buffer 2.1 or buffer 3.1. Select the enzyme and click digest.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Under the Main options header click on &#8220;View gel&#8221; and select the options to run a virtual 1% gel using a pBR322-BstN1 digest.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">Take a screenshot of the results and save this in a text document.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Finally, repeat these steps using both StuI and BsrGI together in a single digest.
</div></li><li data-sub-note-item="2-13"><div class="lab-step">Again, take a screenshot and save the resulting gel.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">DNA Profiling
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Before starting the restriction enzyme digests first put your aliquots of lambda DNA, NEB buffer 2.1, StuI enzyme, and BsrGI enzyme into an ice bucket, and take this to your work area.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Label a tube as StuI &#8211; this will contain the StuI digest. 
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Label a tube as BsrGI to contain the BsrGI digest. 
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Then, label a tube S+B, to contain the digest with both of the enzymes.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Use the appropriate micropipetters to set up the reactions listed in the Table 1:<a href="https://www.jove.com/CDNSource/lm/labs/12/Lab_12_Table_1.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="3-6"><div class="lab-step">First, pipette the 40 &#181;L of water into each tube.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Then, using a fresh pipette tip each time, dispense 5 &#181;L of buffer 2.1 into each tube and pipette up and down to mix.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Next, add 4 &#181;L of lambda DNA to each tube, and then pipette 0.5 &#181;L of each enzyme into the appropriate tubes.
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Finally, add a sufficient amount of water to each tube to bring the total volume up to 50 &#181;L.
</div></li><li data-sub-note-item="3-10"><div class="lab-step">Next, incubate the tubes at 37 &#186;C for 15 - 60 min in a water bath, and return the buffers and enzymes to the freezer.
</div></li><li data-sub-note-item="3-11"><div class="lab-step">To prepare an agarose gel to run the digest, in a 250 mL flask mix 1 g of agarose powder with 100 mL of TAE buffer.
</div></li><li data-sub-note-item="3-12"><div class="lab-step">Microwave the solution for approximately 90 s at full power, taking care to prevent the solution from boiling over.
</div></li><li data-sub-note-item="3-13"><div class="lab-step">Next, carefully remove the solution from the microwave using a glove or heat pad and place it to cool for about 15 min. 
</div></li><li data-sub-note-item="3-14"><div class="lab-step">While the agarose solution cools set up a casting tray. 
</div></li><li data-sub-note-item="3-15"><div class="lab-step">When the agarose solution is cool add 5 &#181;L of 10,000X SYBR Safe gel stain to the solution.
</div></li><li data-sub-note-item="3-16"><div class="lab-step">Add the comb which is used to make the wells of the gel to the casting tray of the gel electrophoresis apparatus.
</div></li><li data-sub-note-item="3-17"><div class="lab-step">Pour the agarose gel solution into the casting tray and allow it to solidify. 
</div></li><li data-sub-note-item="3-18"><div class="lab-step">Once the gel is solid, pull the comb straight up to remove it from the gel, and then remove the dams from the casting tray if appropriate.
</div></li><li data-sub-note-item="3-19"><div class="lab-step">Place the gel in the electrophoresis chamber with the wells positioned closest to the negative or black electrode and fill the chamber with 1x TAE buffer to completely cover the gel.
</div></li><li data-sub-note-item="3-20"><div class="lab-step">Remove your samples from the water bath.
</div></li><li data-sub-note-item="3-21"><div class="lab-step">Then add 8.3 &#181;L of 6X loading dye to each sample.
</div></li><li data-sub-note-item="3-22"><div class="lab-step">Dispense 10 &#181;L of the pBR322-BstN1 digest which contains DNA fragments of known size into the first well.
</div></li><li data-sub-note-item="3-23"><div class="lab-step">Then load 25 &#181;L of sample StuI into the second well.
</div></li><li data-sub-note-item="3-24"><div class="lab-step">Place 25 &#181;L of sample BsRGI into the third well.
</div></li><li data-sub-note-item="3-25"><div class="lab-step">Load 25 &#181;L of sample S+B into the fourth well.
</div></li><li data-sub-note-item="3-26"><div class="lab-step">To run the gel plug the leads into the electrophoresis chamber and then into the power supply. The red positive lead should be at the end farthest away from the wells and the black negative lead should be nearest to the wells. This is because the samples will run from negative to positive.
</div></li><li data-sub-note-item="3-27"><div class="lab-step">Turn on the power supply and set the gel box to run at 170 volts for approximately 60 minutes.
</div></li><li data-sub-note-item="3-28"><div class="lab-step">After the run is complete turn off the power to the gel box and disconnect the electrodes from the tank.
</div></li><li data-sub-note-item="3-29"><div class="lab-step">Then remove the gel from the electrophoresis chamber and observe it with a UV transilluminator.  CAUTION: Protect eyes from the UV by wearing goggles or using the provided shield.
</div></li><li data-sub-note-item="3-30"><div class="lab-step">Finally, use a cell phone camera to take a picture of the gel from directly above and parallel to the gel surface.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">Compare the gel you ran in the lab to your virtual enzymatic digest with StuI.
</div></li><li data-sub-note-item="4-2"><div class="lab-step">The virtual digest with StuI resulted in seven bands. Count the bands in the StuI lane of your gel and note whether this matched the virtual digest or appeared to show a similar pattern. NOTE: Small bands on agarose electrophoresis gels are often invisible.
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Now look at the results for your BsrGI digest. The virtual digest with BsrGI resulted in six bands. Count the bands in the BsrGI lane. Again, note whether the virtual digest and lab digest showed the same or a similar pattern.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Finally, look at the virtual digest with StuI and BsrGI. The virtual digest with both enzymes resulted in 12 bands. Count the bands in the corresponding lane of your gel, and consider whether the DNA banding pattern looks similar to the virtual digest.
</div></li></ul></li></ol>

DNA 分离和限制性内切酶分析

DNA Isolation
ExpandNOTE: In this experiment you will perform DNA isolation under two experimental conditions: one using a buffer containing the detergent ...