<p class="jove_content"> Esta pesquisa foi parcialmente financiado pela National Science Foundation (NSF 1227359), WVU programa EPSCoR financiado pela National Science Foundation (EPS-1003907), WVU escritório ADVANCE patrocinado pela National Science Foundation (1.007.978), e WVU PSCoR, respectivamente. O trabalho foi feito em microfabricação WVU compartilhados Instalações de pesquisa (instalações de salas limpas) e Microfluidic Integrative Research celular no Laboratório Chip (microchip Lab) na West Virginia University. A imagem confocal foi feito em WVU Microscópio Facility Imaging. </p>

<p class="jove_title"> 1. Fotolitografia Fabricação de Moldes Mestre Photoresist </p><p class="jove_content"> O protocolo seguinte ilustra o processo para fabricar os microcanais com diâmetros entre 30-60 um. Para obter um microcanal com um diâmetro mais pequeno (inferior a 30 um), numa única rotação de revestimento de material fotosensitivo é necessário. </p><ol><li> Transferir o material fotosensitivo de refluxo do frigorífico a 4 ° C e a sala limpa 24 horas antes da utilização e permitir que aqueça até à temperatura ambiente. </li><li> Limpar uma bolacha de silício e cozê-la durante uma hora a 150 ° C, para permitir que a desidratar. A desidratação irá ajudar a adesão fotossensível à base de silício. </li><li> Spin-revestir a primeira camada de material fotosensitivo utilizando a seguinte receita: </li></ol><table border="1"><tbody><tr><td> <strong>Passo</strong> </td><td> <strong>Tempo (segundos)</strong> </td><td> <strong>Velocidade (rpm)</strong> </td><td> <strong>Aceleração</strong> </td></tr><tr><td> 1 </td><td> 2 &lt;/ Td&gt; <td> 300 </td><td> maior </td></tr><tr><td> 2 </td><td> 10 </td><td> 0 </td><td></td></tr><tr><td> 3 </td><td> 3 </td><td> 300 </td><td> maior </td></tr><tr><td> 4 </td><td> 60 </td><td> 600 </td><td> maior </td></tr><tr><td> 5 </td><td> 10 </td><td> 500 </td><td> maior </td></tr><tr><td> 6 </td><td> 10 </td><td> 600 </td><td> maior </td></tr></tbody></table><ol start="4"><li> Em seguida, coze a bolacha numa placa de aquecimento com uma temperatura de 110 ° C durante 90 seg. Após a cozer suave da espessura do material fotosensitivo será de 20-30 ^ m. </li><li> Girar revestimento de uma segunda camada de material fotosensitivo seguindo a mesma receita utilizada para a primeira camada. </li><li> Assar a bolacha Mole novamente, colocando-o numa placa de aquecimento com uma temperatura de 110 ° C durante 90 seg. Após este coza suave da espessura do material fotosensitivo será de 40-60 ^ m. </li><li> Gerar padrões de mestre positivos expondo o photoresist à luz UV com uma dose de exposição de 14.500 mJ / cm <sup>2,</sup> por meio de uma máscara de filme. </li><li> Dilui-se com o desenvolvedor desionizada (DI), água (1:2 (v / v)). Lavar a bolacha repetidamente na solução até que o padrão está completamente desenvolvido. Em seguida, lave o wafer com água DI e seque-o com gás nitrogênio. </li><li> Refluxo: Coloque a bolacha numa placa de aquecimento com uma temperatura de 120 ° C durante 4 min e com uma tampa de uma placa de Petri de vidro para evitar a evaporação do solvente. Retire o wafer do fogão e deixe esfriar à temperatura ambiente. A espessura do material fotosensitivo após o refluxo será de 50-60 ^ m. </li></ol><p class="jove_title"> 2. Fabricação de litografia suave de PDMS Microcanal Rede </p><ol><li> Preparar a solução de polidimetilsiloxano (PDMS) com a proporção em peso de 10:01 (base: agente de cura) e misture bem utilizando um misturador planetário centrífuga. </li><li> Lançai a solução PDMS para o molde mestre photoresist refluído. Colocar o PDMS fundido num exsicador durante 15 min para desgaseificar. Use azoto gasoso para remover quaisquer bolhas restantes, se necessário. </li><li> Asse o PDMS numa estufa a uma temperatura de 60 ° C durante 3 horas para permitir a cura. Em seguida, retire a camada de PDMS curada do molde mestre. </li><li> É um perfurador aguçado para criar os orifícios de entrada / saída por furos de perfuração na rede de canais. Limpar a superfície do PDMS usando gás azoto. </li><li> Trate duas camadas de PDMS com plasma de oxigênio por 30 segundos dentro de um aspirador de plasma a uma pressão de operação de 4,5 x 10 <sup>-2</sup> Torr e uma taxa de fluxo de oxigênio de 3,5 pés <sup>3</sup> / min. Em seguida, alinhar as superfícies do PDMS manualmente sob um microscópio óptico. Use uma gota de água se necessário, para um melhor controlo do alinhamento. </li><li> Asse o dispositivo num forno a 60 ° C durante 30 min para conseguir a ligação permanente. </li></ol><p class="jove_title"> 3. Cultura HUVEC e propagação no Chip </p><ol><li> Cultura das HUVECs com meio de cultura com L-glutamina, suplementado com 10% de soro fetal bovino (FBS). Por diaforam usadas passagens electrónicos experimentais entre 2 e 5. </li><li> Uma vez que as HUVECs são confluentes, a contagem das células e colhem-lhes, em primeiro lugar a lavagem das células com solução salina tamponada com HEPES (HEPES-BSS) e, em seguida, tratar as células com tripsina / EDTA e incubar durante 2-6 min a 37 ° C. Após a tripsinização é completa, neutralizar a tripsina / EDTA com uma solução de neutralização de tripsina. Centrifugar e suspender as células em meio de cultura com 8% de dextrano para recolhê-las. Dextrano foi usada para aumentar a viscosidade média para ajudar na melhor semeadura de células e do acessório. </li><li> Tratar o dispositivo com plasma de oxigénio durante 5 min com uma pressão de serviço de 4,5 x 10 <sup>-2</sup> Torr e uma taxa de fluxo de oxigénio de 3,5 ft <sup>3</sup> / min. Em seguida, carregar o dispositivo com água DI e tratar com luz UV por 8 horas em um laminar capa de biossegurança para a esterilização. </li><li> Um dia antes das células estão prontas, o dispositivo de lavagem com 1 x tampão fosfato salino (PBS 1x) e depois o revestimento com fibronectina (100 ug / ml, dilbuído com PBS 1x) e incubar no frigorífico a 4 ° C durante a noite. </li><li> Depois do revestimento com a fibronectina, o dispositivo de lavagem com 1x PBS, em seguida, carregar com meio de cultura. Incubar o dispositivo a uma temperatura de 37 ° C durante 15 min. </li><li> Carregar as células HUVEC em meio de cultura de 8% de dextrano com uma concentração celular de 3-4 x 10 <sup>6</sup> células / ml. Coloque uma gota de 20 ul de células a uma entrada do dispositivo e inclinar-se a introdução das células no interior do canal microfluídico. Depois de 15-20 minutos as células começarão a anexar as paredes laterais dos canais. Girar o dispositivo a cada 15 minutos para criar uma distribuição mais uniforme das células. Se necessário, uma carga adicional pode ser executada. </li></ol><p class="jove_title"> 4. Configuração de perfusão de longo prazo </p><p class="jove_content"> Depois de 5-6 horas de cultura estática, os HUVECs anexados vai começar a espalhar totalmente. Configure perfusão usando um sistema de bomba de seringa controle remoto com um fluxo constante de 10 mL / h. A perfusão pode ser ajustada fou uma taxa de fluxo mais elevada, e pode durar por um período de tempo entre 4 dias a 2 semanas. </p><p class="jove_title"> 5. Coloração celular e Caracterização Microscópio </p><ol><li> Quando as células atingem confluência no interior do dispositivo, em primeiro lugar, o dispositivo de lavagem com 1x PBS para remover completamente o meio. Em seguida, carregar o aparelho com corante vermelho diluído com diluente (4 ul-1 ml). Carregar o corante semelhante ao procedimento de carregamento da célula. Incubar o dispositivo no escuro durante 5 min à temperatura ambiente, e, em seguida, o dispositivo de lavagem com meio de cultura para parar a coloração. Comprida incubação do corante pode causar toxicidade celular e disadhesion. </li><li> Carregar o dispositivo com o corante azul diluída com PBS 1x (2 gotas por ml). Incubar no escuro durante 5 min à temperatura ambiente e depois lavar com o dispositivo de 1x PBS. </li><li> Examine a coloração de células em microscópio óptico invertido. Se a coloração era bom, carregar os microcanais com meio de fixação (3,5% paraformaldeído diluído com PBS 1x), em seguida submergiro dispositivo de fixação no suporte e cobri-lo completamente com folha de alumínio. Armazenar o dispositivo no frigorífico a uma temperatura de 4 ° C para evitar que o dispositivo de secagem e fotobranqueamento. O dispositivo fixo está agora pronto para imagiologia confocal, o que pode ser feito por um microscópio de varrimento de laser confocal. </li></ol>

<p class="jove_content"> Os autores declaram que não têm interesses financeiros concorrentes. </p>

<html><p class="jove_step"> 1. Fabricação de moldes Mestre </p><p class="jove_content"> Um dos princípios orientadores para a concepção e morfometria vascular é conhecida como lei de Murray, <sup>16,</sup> que afirma que a distribuição dos diâmetros dos vasos em toda a rede é regida por contrapartida mínima de energia. Afirma também que o cubo dos diâmetros de um navio-mãe a uma bifurcação é igual à soma dos cubos dos diâmetros dos vasos filha ( <img alt="Equação 1" fo:content-width="0.9in" fo:src="/files/ftp_upload/50771/5...

<p class="jove_content"> Microvasos, como uma parte do sistema de circulação, medeiam as interacções entre o sangue e os tecidos, suporta as actividades metabólicas, definir microambiente do tecido, e desempenham um papel fundamental em muitos estados patológicos e de saúde. Recapitulação de microvasos funcionais <em>in vitro</em> pode fornecer uma plataforma para o estudo de fenômenos vasculares complexos. No entanto, <em>em</em> ensaios convencionais microvasos <em>in vitro,</em> tais como ensaios de migração de células endoteliais, ensaios de formação de tubos endoteliais e de rato e ensaios de anel da aorta do rato, são incapazes de recriar o microvasos <em>in vivo</em> no que diz respeito à geometria tridimensional (3D) e controlo de fluxo contínuo <sup>1-8.</sup> Estudos de microvasos com modelos animais e <em>em</em> ensaios <em>in vivo,</em> tais como o ensaio de angiogénese da córnea, pinto corioalantóico ensaio de angiogénese da membrana e ensaio de tampão de Matrigel, são mais demorado, alto custo, um desafio no que diz respeito à observação e quantificação, elevantar questões éticas <sup>1, 9-13.</sup> </p><p class="jove_content"> Avanços em Microfabricação e tecnologias de chips microfluídicos têm permitido uma variedade de insights sobre ciências biomédicas, enquanto reduzindo os altos custos experimentais e as complexidades associadas com animais e estudos <em>in vivo</em> <sup>de 14,</sup> como condições biológicas de forma fácil e rigidamente controlada e ambientes fluidos dinâmicos, que não teriam foi possível com as técnicas convencionais macroescala. </p><p class="jove_content"> Aqui, apresentamos uma abordagem para a construção de uma reendotelizados microcanais-on-a-chip que imita a geometria 3D <em>in vivo</em> microvasos e é executado sob fluxo de perfusão contínua controlada usando a combinação da técnica de fotolitografia reflowable photoresist, litografia macia e microfluídica. </p>

<table><tbody><tr><td>&lt;strong&gt;Reagent/Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Reflow Photoresist</td><td>AZ Electronic Materials</td><td>AZP4620</td><td></td></tr><tr><td>Developer</td><td>AZ Electronic Materials</td><td>AZ 400K</td><td></td></tr><tr><td>PDMS</td><td>Dow Corning Corporation</td><td>Sylgard 184</td><td></td></tr><tr><td>MCDB 131 Culture Medium</td><td>Invitrogen</td><td>10372-019</td><td></td></tr><tr><td>NacBlue Nuclei Staining</td><td>Invitrogen</td><td>H1399</td><td></td></tr><tr><td>PKH Red Stain</td><td>Sigma</td><td>MINI26 and PKH26GL</td><td></td></tr><tr><td>Fibronectin</td><td>Gibco</td><td>PHE0023</td><td></td></tr><tr><td>L-Glutamine</td><td>Sigma</td><td>G7513</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Invitrogen</td><td>14040-133</td><td></td></tr><tr><td>HEPES Buffered Saline Solution</td><td>Lonza</td><td>CC-5024</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Invitrogen</td><td>25300-062</td><td></td></tr><tr><td>Trypsin Neutralizing Solution</td><td>Lonza</td><td>CC-5002</td><td></td></tr><tr><td>PDMS Curing Agent</td><td>Dow Corning Corporation</td><td>Sylgard 184</td><td></td></tr><tr><td>Primary Human Umbilical Vein Endothelial Cells</td><td>Lonza</td><td>CC-2517</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Lonza</td><td>14-501F</td><td></td></tr><tr><td>Diluent C</td><td>Sigma</td><td>CGLDIL</td><td></td></tr><tr><td>Hoechst33342</td><td>Invitrogen, Molecular Probes</td><td>R37605</td><td></td></tr><tr><td>Dextran</td><td>Sigma</td><td>95771</td><td></td></tr><tr><td>3.5% Paraformaldehyde</td><td>Electron Microscopy Science</td><td>15710-S</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Spinner</td><td>Laurell Technologies Corporation</td><td>WS-400BZ-6NPP/LITE</td><td></td></tr><tr><td>Desiccator</td><td>BelArt Products</td><td>999320237</td><td></td></tr><tr><td>Inverted Microscope</td><td>Nikon</td><td>Eclipse Ti</td><td></td></tr><tr><td>Syringe Pump System</td><td>Harvard Apparatus</td><td>PHD Ultra</td><td></td></tr><tr><td>Laminar Biosafety Hood</td><td>Thermo Scientific</td><td>1300 Series A2</td><td></td></tr><tr><td>Planetary Centrifugal Mixer</td><td>Thinky</td><td>ARE-310</td><td></td></tr><tr><td>Isotemp Oven</td><td>Fisher Scientific</td><td>13-246-516GAQ</td><td></td></tr><tr><td>Optical Microscope</td><td>Zeiss</td><td>Invertoskop 40C</td><td></td></tr><tr><td>Plasma Cleaner</td><td>Harrick Plasma</td><td>PDC-32G</td><td></td></tr><tr><td>Hotplate</td><td>Barnstead/Thermolyne Cimarec</td><td>SP131635</td><td></td></tr><tr><td>Laser Scanning Confocal Microscope</td><td>Zeiss</td><td>LSM 510</td><td></td></tr></tbody></table>

procedure for the development of multi-depth circular cross-sectional endothelialized microchannels-on-a-chip

<p class="jove_content"> Os esforços têm sido focados no desenvolvimento de ensaios <em>in vitro</em> para o estudo de microvasos porque estudos em animais <em>in vivo</em> é mais demorado, caro e de observação e quantificação são muito desafiante. No entanto, <em>em</em> ensaios <em>in vitro</em> convencional microvasos têm limitações quando representar microvasos <em>in vivo</em> no que diz respeito a geometria tridimensional (3D) e fornecer o fluxo de fluido contínuo. Usando uma combinação de técnicas de fotolitografia reflowable photoresist, litografia macia e microfluídica, temos desenvolvido um multi-profundidade reendotelizados transversais circulares microcanais-on-a-chip, que imita a geometria 3D <em>in vivo</em> microvasos e é executado sob perfusão contínua controlada fluxo. Um fotosensitivo refluído positivo foi usada para fabricar um molde mestre, com uma rede de microcanais transversal semicircular. No alinhamento e união das duas (PDMS) microcanais polidimetilsiloxano replicated do molde mestre, uma rede de microcanais cilíndrico foi criado. Os diâmetros dos microcanais pode ser bem controlada. Além disso, as células primárias endoteliais da veia umbilical humana (HUVEC) semeadas dentro do chip mostraram que as células revestidas a superfície interior dos microcanais em perfusão controlada duradouro durante um período de tempo entre 4 dias a 2 semanas. </p>

<p class="jove_content"> A nossa abordagem para fabricar a rede de microcanais múltiplos profundidade imita as geometrias complexas 3D de microvasos <em>in vivo,</em> em que os microcanais têm secções transversais arredondadas <sup>15.</sup> Além disso, os diâmetros dos canais de ramificação-mãe e os canais filha aproximadamente obedecem à lei de Murray, para manter o fluxo do fluido em um nível requerido de modo que a resistência global do canal é baixa e velocidades de fluxo são mais uniforme em toda a rede de <sup>16-18.</sup> ...

Watch this Scientific Journal Video about Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip at JoVE.com

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

<p class="jove_content"> Uma plataforma de microcanais-on-a-chip foi desenvolvido pela combinação da técnica fotolitográfica refluído fotossensível, litografia macia, e microfluidos. A plataforma de microcanais reendotelizados imita a geometria tridimensional (3D) de microvasos <em>in vivo,</em> é executado sob fluxo de perfusão contínua controlada, permite a imagem de alta qualidade e em tempo real, e pode ser aplicado para a pesquisa microvascular. </p>

Procedimento para o Desenvolvimento da Multi-profundidade Circular transversal reendotelizados microcanais-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

procedure-for-development-multi-depth-circular-cross-sectional

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

13.8K visualizações.  West Virginia University.  Uma plataforma de microcanais-on-a-chip foi desenvolvido pela combinação da técnica fotolitográfica refluído fotossensível, litografia macia, e microfluidos. A plataforma de microcanais reendotelizados imita a geometria tridimensional (3D) de microvasos in vivo, é executado sob fluxo de perfusão contínua controlada, permite a imagem de alta qualidade e em tempo real, e pode ser aplicado para a pesquisa microvascular. 

Vídeo: Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Galvanic Cells

<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</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare 0.1 M oxalic acid dihydrate. Each group will need 50 mL. For 500 mL of solution, weigh 6.304 g of oxalic acid, and transfer it to an Erlenmeyer flask. <strong>Note:</strong> Oxalic acid is corrosive, so use caution while handling.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Measure 500 mL of deionized water, and pour it into the flask. Add a stir bar and set the flask onto a stir plate. Turn on the stir setting and allow the solution to mix until it is homogeneous.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Obtain a polyethylene bottle with a cap and label it. Use a powder funnel to transfer the solution to the bottle, cap it, and store it in the instructor&#39;s hood until the lab.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Prepare a 0.1 M potassium nitrate solution, which will be used to soak the strings for the salt bridge. To make 250 mL of solution, weigh 2.53 g of potassium nitrate and transfer it to a flask.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Add 250 mL of deionized water to the flask and stir the solution until it appears homogeneous.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Transfer the solution into a labeled glass bottle and store it in the instructor&#39;s hood until the lab class.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Prepare a 0.05 M zinc sulfate solution, which will be used as an unknown solution in the experiment. Each group will need 8 mL of solution, so calculate how much you will need to make for your class.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">To make 250 mL of solution, weigh 3.6 g of zinc sulfate and transfer it to an Erlenmeyer flask.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Add 250 mL of deionized water to the flask and set the solution on the stir plate to mix.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Once the solution appears homogeneous, divide it into two brown glass bottles.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Label one of the bottles as &#8216;unknown number 1&#8217;, and the other as &#8216;unknown number 3&#8217;. Store the bottles in the instructor&#39;s hood until the lab.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Prepare 0.22 M silver nitrate, which will also be used as an unknown solution in the experiment. Each group will need 8 mL. To prepare 250 mL of solution, weigh 9.34 g of silver nitrate, and transfer it to the flask.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Add 250 mL of deionized water and mix the solution until it is homogeneous.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Then, transfer the solution to two brown glass bottles. Label them &#8216;unknown number 2&#8217; and &#8216;unknown number 4&#8217;. Store the bottles with the other prepared solutions in the instructor&#39;s hood.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Preparation of Other Reagents<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Each group will need about 1.25 g of copper sulfate. Place the bottle next to the analytical balance along with a clean spatula and weigh boats.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Each group will need about 0.35 g of lead nitrate. Place the bottle next to a balance.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">You&#39;ll also need 1 x 9 cm pieces of copper sheet. Count out enough for each group to have two pieces and place them in a container.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Each group will also need one piece of lead. Obtain 2 x 6 cm pieces of lead and put them in a labeled beaker.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Obtain 2 x 6 cm pre-cut pieces of zinc sheet, one for each group. Divide the strips into two beakers with one labeled as unknown number 1 and the other as unknown number 3.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Each group will also need one 2 x 6 in piece of silver sheet. Divide the strips into two beakers with one labeled as &#8216;unknown number 2&#8217;, and the other as &#8216;unknown number 4&#8217;.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Cut emery paper in 1 x 2 in pieces so that each group has two pieces.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Cut filter paper into 1 x 9 in pieces with one for each group.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Cut pieces of string 2 in long. Each group will need three pieces of string, so cut as many as are needed for the class size.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">An hour before the lab class begins, place the pieces of string in the bottle of potassium nitrate and allow them to soak.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Ensure that a 20-L container is dedicated to heavy metal waste and is accessible for all students in the hood.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">Set out the remaining 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;Multimeter with red and black leads</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Alligator clips</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Glass thermometer</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;6-well reaction plate</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Pair of forceps</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;20-mL volumetric flask</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;100-mL volumetric flask</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;1-mL volumetric pipette</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;2-mL capacity pipettor</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;3&#160;&#160;&#160;&#160;100-mL beakers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;10-mL graduated cylinder</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;1&#160;&#160;&#160;&#160;600-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Glass funnels</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
	</ul>
	</li>
</ol>

Galvanic Cells: Construction and Reduction Potential Measurement - Prep

Células Galvânicas

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

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

Education

JoVE Lab Manual

Química

Lab Manual Sub Articles

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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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

Células eletrolíticas

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

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

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

Cromatografia em camada fina

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

	Effects of Mobile Phase Composition on Compound ...

Column Chromatography

<p>Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Column Chromatography of Food Dye<button class="expand">Expand</button>
	<p>In this experiment, you&#39;ll use column chromatography to separate green food coloring into its component blue and yellow dyes, called erioglaucine and tartrazine. First, you&#39;ll use 95% ethanol to elute the dye with a weaker affinity for silica gel. Then, you&#39;ll flush the second dye from the column with water. To recover the purified dyes, you will collect each band of dye as it leaves the column. These collected volumes are called fractions.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">To begin, put on a lab coat, safety glasses, and a pair of nitrile gloves.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Bring a column or burette to the central supply area. Obtain a piece of cotton or glass wool and press it into the top of your column. Use a long, blunt-ended wire or rod to push it down to plug the column just above the stopcock.<strong> Note: </strong>The cotton must be stable, but don&#39;t pack it too densely, because the solvent needs to pass through it easily. Make sure that the stopcock can turn freely without catching on the plug.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Attach a burette clamp to a lab stand and clamp the column upright with about 4 in of space underneath it. Make sure that the stopcock is closed.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Label a 100-mL beaker as &#8216;waste&#8217; and place it under the column.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Next, measure 1 g of sand in a tared weighing boat and bring it back to the bench.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Then, mark the column about 0.5 cm above the top of the cotton plug.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Place a small funnel in the top of the column and pour enough sand through the funnel so that there is a layer 0.5 cm deep over the plug. This will keep the silica gel from passing through the plug into your fractions.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Remove the funnel and gently tap the column to evenly distribute the sand.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Now, prepare your eluents. Label a 50-mL beaker and a 50-mL graduated cylinder as &#8216;water&#8217;.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Measure 50 mL of deionized water with the graduated cylinder and pour it into the beaker.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Then, label another 50-mL beaker and a 50-mL graduated cylinder as &#8216;ethanol&#8217;. Measure 50 mL of 95% ethanol and pour it into this beaker.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Now, obtain 4 or 5 clean Pasteur pipettes and a pipette bulb. Gently pipette 7 &#8211; 8 mL of ethanol along the inner walls of the column. This layer of solvent will let you pour this silica gel slurry into the column without disturbing the sand.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Open the stopcock and drain ~1 mL of ethanol into the waste beaker to flush air from the stopcock and to confirm that the solvent flows easily through the plug. Make sure that there are no dry patches in the sand and the plug and gently tap the side of the column to remove trapped air bubbles.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Next, place a clean funnel in the top of the column and label a third 50-mL beaker &#8216;silica gel slurry in ethanol&#8217;.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Carefully weigh 5 g of silica gel powder and pour it into your labeled beaker.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Then, bring the silica gel, a glass rod, and your ethanol graduated cylinder to the ethanol dispensing hood.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Measure 18 mL of 95% ethanol and pour it into the beaker. Stir the mixture with the glass rod until the texture is consistent and no dry patches or clumps of powder remain. This usually takes ~1 min. <strong>Note: </strong>The slurry should be thick, like batter, but it should still flow easily. If the slurry is too stiff, it could clog the funnel or trap air bubbles in the column.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Once the powder is completely suspended in ethanol, bring it to your bench. Stir the slurry until its texture is consistent again and immediately pour it into the column.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Gently tap the sides of the column as the silica gel settles to get rid of air bubbles.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Rinse the beaker with 1 or 2 mL of ethanol, and pour the rinse into the column. Then, remove the funnel and set it aside.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Pipette ethanol along the inside of the column to wash down particles stuck to the walls. Then, drain the solvent until the meniscus is just barely above the top of the silica gel.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Now, use a 5- or 10-mL graduated cylinder to obtain about 0.8 mL of the green food dye solution.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Use a clean pipette to gently apply 5 to 7 drops of the dye solution evenly across the top of the silica gel. If necessary, add a few drops of ethanol to keep the silica gel covered.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">While you wait for the dye to soak in, label two clean 50-mL beakers as &#8216;ethanol fraction&#8216; and &#8216;water fraction&#8217;. Exchange the waste beaker under the column with the ethanol fraction beaker.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Next, fill the column with ethanol, pipetting the first mL along the inner walls to avoid disturbing the silica gel.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Then, open the stopcock partway and adjust it until the solvent level decreases ~1 mL every 15 &#8211; 20 s.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Periodically refill the column to keep the ethanol flowing. Make sure that there is at least 1 cm of solvent above the silica gel at all times.</div>
		</li>
		<li data-sub-note-item="1-29">
		<div class="lab-step">Monitor the yellow and blue bands as they separate. <strong>Note: </strong>If you need to step away, close the stopcock so that the column won&#39;t run dry while you&#39;re gone. Avoid stopping for more than 1 min at a time because the compound bands will start broadening. It usually takes about 20 &#8211; 35 min for the faster-moving dye to leave the column.</div>
		</li>
		<li data-sub-note-item="1-30">
		<div class="lab-step">Once you have collected all of the faster-moving dye in the ethanol fraction beaker, close the stopcock, set the ethanol fraction aside, and put the waste beaker under the column.</div>
		</li>
		<li data-sub-note-item="1-31">
		<div class="lab-step">Drain the solvent until the meniscus is just above the top of the silica gel. Then, exchange the waste beaker for the water fraction beaker.</div>
		</li>
		<li data-sub-note-item="1-32">
		<div class="lab-step">Use a clean pipette to fill the top of the column with deionized water. Then, open the stopcock just enough to let water flow through the column at a rate of about 1 mL every 15 &#8211; 20 s.</div>
		</li>
		<li data-sub-note-item="1-33">
		<div class="lab-step">Refill the column with water as needed until the second band of color has completely left the column. This usually takes another 15 &#8211; 25 min. Then, close the stopcock and exchange the water fraction beaker for the waste beaker.</div>
		</li>
		<li data-sub-note-item="1-34">
		<div class="lab-step">Now, drain the column completely and leave the stopcock open.</div>
		</li>
		<li data-sub-note-item="1-35">
		<div class="lab-step">Record the colors of the ethanol and water fractions in your lab notebook.
		<h4><strong>Table 1: Column Chromatography of Food Dye</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td></td>
					<td><strong>Color </strong></td>
					<td><strong>Volume (mL) </strong></td>
				</tr>
				<tr>
					<td>Ethanol fraction</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Water fraction</td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/63/Table_1._Column_chromatography_of_food_dye.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-36">
		<div class="lab-step">Pour the fractions into the corresponding 50 mL graduated cylinders and record the volume of each fraction. Then, close the stopcock and bring the column to a fume hood.</div>
		</li>
		<li data-sub-note-item="1-37">
		<div class="lab-step">Clamp the column upright in the hood so that the silica gel can dry. Your instructor will either tell you how to clean the column or take care of it after the lab.</div>
		</li>
		<li data-sub-note-item="1-38">
		<div class="lab-step">Now, flush the collected fractions and leftover ethanol, water, and dye solution down the drain and clean the rest of your glassware as usual.</div>
		</li>
		<li data-sub-note-item="1-39">
		<div class="lab-step">Discard used Pasteur pipettes in the glass waste. Throw out used paper towels and weighing boat in the lab trash and clean the benchtop before you leave.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Results<button class="expand">Expand</button>
	<p>In this experiment, you saw that ethanol eluted the blue erioglaucine in a timely manner, but it wasn&#39;t an effective eluent for the yellow tartrazine. Paper chromatography of the green dye shows similar results. Thus, we conclude that erioglaucine has a lower affinity for silica gel than tartrazine does.</p>
	<p>Now, consider the advantages of using two solvents to elute the dye separately. Water is more polar than ethanol, so we assume that it would elute both dyes faster than ethanol alone would. While you also would have used less solvent overall if you had only used water, the separation would have been worse, making it harder to collect pure fractions.</p>
	<p>Alternatively, you could have kept using ethanol after you collected the erioglaucine, but it would have taken a long time and a lot of ethanol to elute tartrazine that way. Switching the eluent to water after eluting erioglaucine saved time and minimized solvent waste without sacrificing purity.</p>
	<ul class="lab-items">
	</ul>
	</li>
</ol>

Column Chromatography: Experimental Setup and Separation - Procedure

Cromatografia em coluna

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

	Column Chromatography of Food DyeExpand
	In this ...

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

Estrutura Celular

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

Biologia

Fundamentais

Community Diversity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Measuring Biodiversity in Areas with Different Disturbance Regimes
<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 compare the biodiversity in a less disturbed area against a more highly disturbed area. HYPOTHESES: The experimental hypothesis could be that a less disturbed area will contain a greater diversity of species than a highly disturbed area. The null hypothesis could be that there will be no difference in diversity between less disturbed and highly disturbed areas. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To begin, divide into groups of 2-4 and collect a hula hoop, ball, measuring tape, set of printed tables, 10 utility flags, and an ID guide from the front of the class.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Additionally, collect a shovel and 10 collecting bottles for the soil sampling exercise.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Once in the field, your instructor will show you the two habitats you will study - choose one habitat to begin.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Place one flag in each of four corners to form a rectangular sampling area.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Measure the length and width of the sampling area in meters, and then multiply the two numbers to determine the area.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Standing in the middle of the sampling area, throw a tennis ball into the air, and then Mark the location where it lands with a flag.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Repeat this random throw four additional times to determine all five of your random sampling points.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Keeping your flag at the center, place a hula hoop around it. The area within the hula hoop is a quadrat.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Identify all of the different species in the quadrat and record them as species A, B, and so on in Table 1, provided by your instructor.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Once all of the plants have been recorded, use a shovel to collect one cup of soil from each of the sites and put the samples bottles labeled with your names and the site name and quadrat number.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Repeat the plant identification and soil sampling for the remaining four quadrats in the first habitat, recording your observations in Table 1.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Once you have completed recording in the first habitat, move on to the second field, again marking out a rectangular sampling area.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Repeat the random placement of quadrats using the tennis ball, and then record the species present in each quadrat in Table 2. 
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Finally, collect one cup of soil from each of the disturbed habitats&#39; quadrats, for a total of five cups from the disturbed and five cups from the undisturbed habitats.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Measuring Biodiversity Along a Transect
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">NOTE: The next part of the study will test for edge effects, or a change along a gradient from where one ecosystem type meets another to the core habitat of the ecosystem. HYPOTHESES: The experimental hypothesis is that there will be a greater diversity of species closer to the edge of the ecosystem than in the center, or core, of the ecosystem. The null hypothesis is that there is no change in diversity along the transect from edge to core habitat.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Place a flag at the perimeter of the field where it meets another habitat, like a forest or parking lot.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Extend a tape measure towards the center of the field, perpendicular to the edge of the field. This will be the transect.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Place a flag at the zero meter of the transect (the edge of the other habitat) then place additional flags at 10 m, 20 m, 30 m, and 40 m into the core of the habitat for a total of five flagged locations.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Starting at the 0 m point along the transect, place the hula hoop at the flagged location. Record the species present in the quadrat in Table 4.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Now repeat the quadrant placement and species recording at the 10 m, 20 m, 30 m, and 40 m marks.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Next, place two more transects from different starting points at the perimeter of the field extending into the core area.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Mark the 10-meter points along these transects, and again use these as markers to place quadrats. Record the species found in each of these quadrats in Table 4.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Processing the Soil Samples
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">HYPOTHESES: The experimental hypothesis is that the two landscapes under different disturbance regimes will differ in their soil composition and pH. The null hypothesis is that there will be no difference in the soil composition or pH between the two sites. 
</div></li><li data-sub-note-item="3-2"><div class="lab-step">To begin, remove soil from one bottle of the first site sampled and fill the pH color compartment of the soil sample kit to the soil fill line.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Pour the pH test capsule into the test chamber, then fill the capsule with distilled water until it reaches the water fill line.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Place the cap on the compartment and shake it.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Allow the color to develop for about a minute, and then compare the soil to the color chart to determine the pH.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Repeat the pH analysis for the second sampled site, filling out Table 3 for the recorded pH.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">To process the soil samples for nitrogen, phosphorus, and potassium, remove 20 mL of soil from the bottle of the first site sampled.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Place the soil in a clean container with 100 mL of distilled water.
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Repeat this process for the second site, labeling each container, and then allow them to sit for one hour.
</div></li><li data-sub-note-item="3-10"><div class="lab-step">After one hour, use a dropper to fill each of the nitrogen, phosphorous, and potassium comparators to the fill line with solution from the first sampled site.
</div></li><li data-sub-note-item="3-11"><div class="lab-step">Add the appropriate capsule to each of the comparators for each chemical test.
</div></li><li data-sub-note-item="3-12"><div class="lab-step">Cap and shake the comparators thoroughly, and then compare the solution in the test chamber to the color chart, recording the results as the appropriate number on the scale into Table 3.
</div></li><li data-sub-note-item="3-13"><div class="lab-step">Repeat the nitrogen, phosphorous, and potassium tests for each of the sampled sites. NOTE: Rinse the comparator thoroughly between samples
</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">To calculate the Shannon-Weiner Diversity for each of the observed sites, first open a new Excel spreadsheet.
</div></li><li data-sub-note-item="4-2"><div class="lab-step">Label cell A1 'species name', and cell B1 'total count'.
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Starting from the second row and in column A, fill in the sheet for each species found in the first site, using the data recorded in Table 1.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Calculate the total number of each species found in the entire sampling effort and record this in the B column of the Excel spreadsheet.
</div></li><li data-sub-note-item="4-5"><div class="lab-step">Now, label cell G1 'total count of all species', and in cell H1, write a formula, or highlight the appropriate cells to sum all of the count values entered in column B: = SUM(B2: BX)
</div></li><li data-sub-note-item="4-6"><div class="lab-step">Label cell C1 'proportion of each species' and in cell C2, type the formula for the proportion of the total of each species at the site. = B2/H$1
</div></li><li data-sub-note-item="4-7"><div class="lab-step">Drag this formula down for each row of data represented in column B.
</div></li><li data-sub-note-item="4-8"><div class="lab-step">Label cell D1 'natural log proportion of species' and in cell D2, type the formula for the natural log of the proportion of species at the site. =LN(C2)
</div></li><li data-sub-note-item="4-9"><div class="lab-step">Drag this formula down for each row of data represented in column C.
</div></li><li data-sub-note-item="4-10"><div class="lab-step">Add the formula into cell E1 for the proportion of species multiplied by the natural log of the proportion of species: ps* ln ps
</div></li><li data-sub-note-item="4-11"><div class="lab-step">In cell E2, type the formula multiplying the proportion of species by the natural log of the proportion of species at each site, and then drag this formula down for each row of data represented in column D. = C2 * D2 
</div></li><li data-sub-note-item="4-12"><div class="lab-step">Label cell G2 as 'sum of ps * ln ps', and then, in cell H2, type a formula summing all of the values in column E. = SUM(E3 - EX) 
</div></li><li data-sub-note-item="4-13"><div class="lab-step">Label cell G3 'Shannon-Weiner Index', and in cell H3, enter the formula for the Shannon-Weiner Diversity Index. = (EXP - 1 * H2) NOTE: This is the calculated Shannon-Weiner Diversity Index for the first sampled site, and will be used for comparison between the two sites.
</div></li><li data-sub-note-item="4-14"><div class="lab-step">Repeat the data analysis in a new sheet using the data collected for the second site, which was recorded in Table 2. NOTE: Areas with greater index values are considered to have greater species diversity. Note whether you see differences between your sites.
</div></li><li data-sub-note-item="4-15"><div class="lab-step">Repeat the Shannon-Weiner Diversity calculations using a total count of all the species at each distance along the transect, then create a table of all of these values in a new Excel sheet.
</div></li><li data-sub-note-item="4-16"><div class="lab-step">Click on the Data tab in Excel, and then on the 'Data Analysis' button.
</div></li><li data-sub-note-item="4-17"><div class="lab-step">Select 'Regression' and click 'Okay'.
</div></li><li data-sub-note-item="4-18"><div class="lab-step">For the Input Y Range, highlight the Shannon-Weiner Diversity values, and for the Input X range, highlight the distance values.
</div></li><li data-sub-note-item="4-19"><div class="lab-step">Select an output location for the regression results and click 'Okay'.
</div></li><li data-sub-note-item="4-20"><div class="lab-step">Check the P-value box in the intercept row to see if there is a significant correlation between distance from edge to core habitat and biodiversity. NOTE: A value of less than 0.05 rejects the null hypothesis of no change in diversity along the transect, while a value greater than 0.05 does not reject the null hypothesis.
</div></li><li data-sub-note-item="4-21"><div class="lab-step">Next, to perform t-tests for determining soil composition differences between sites, open a new Excel spreadsheet.
</div></li><li data-sub-note-item="4-22"><div class="lab-step">In cell A1, type 'Site 1:pH', and in cell B1, type 'Site 2:pH'.
</div></li><li data-sub-note-item="4-23"><div class="lab-step">Fill in cells A2 through A6 and cells B2 through B6 with the soil pH collected in the disturbed and undisturbed sites, respectively.
</div></li><li data-sub-note-item="4-24"><div class="lab-step">Click the Data tab in Excel and then select 'Data Analysis'.
</div></li><li data-sub-note-item="4-25"><div class="lab-step">Now scroll down and select 't-Test: Two-Sample Assuming Equal Variances' and select 'Okay'.
</div></li><li data-sub-note-item="4-26"><div class="lab-step">Click the drop down next to Variable 1 Range and highlight cells A2 through A6 in the spreadsheet, then click the dropdown next to Variable 2 Range and highlight cells B2 through B6 in the spreadsheet.
</div></li><li data-sub-note-item="4-27"><div class="lab-step">In the box for hypothesized mean difference, type zero as our null hypothesis. This means that there is no difference between the two disturbance regimes.
</div></li><li data-sub-note-item="4-28"><div class="lab-step">Leave the Labels box unchecked and set the alpha value to 0.05.
</div></li><li data-sub-note-item="4-29"><div class="lab-step">Select 'Output Range', and in the box, click an empty space in the spreadsheet.
</div></li><li data-sub-note-item="4-30"><div class="lab-step">Click 'Okay'. A table of t-Test results should be outputted in the spreadsheet.
</div></li><li data-sub-note-item="4-31"><div class="lab-step">The relevant data are the means for each of the sites and the alpha value. If this second value is less than our alpha value of 0.05, we can reject the null hypothesis that there is no difference in pH between the two sites, and the hypothesis that disturbed versus undisturbed sites differ in their pH value would gain support. If this value is greater than 0.05, however, we may not reject our null hypothesis.
</div></li><li data-sub-note-item="4-32"><div class="lab-step">Repeat this analysis for each of the other three soil variables, creating a new spreadsheet for each of the phosphorus, potassium, and nitrogen elements. 
</div></li><li data-sub-note-item="4-33"><div class="lab-step">Note whether your pH and nutrient level results were different in each condition, and discuss with your group how this might affect the community and diversity in the different sites.
</div></li></ul></li></ol>

Community Diversity: Measuring Biodiversity in Different Areas - Procedure

Diversidade da comunidade

Measuring Biodiversity in Areas with Different Disturbance Regimes
ExpandNOTE: In this experiment, you will compare the biodiversity in a less disturbed ...

Ecologia

Cell Division

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Preparation of Onion Root Tips and Solutions
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">For the cell cycle experiment using root tips, first, leave an onion suspended over a beaker of water to grow roots for several days. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Then, clean the onion roots of any dirt or debris. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Next, in a 1.5 mL tube, dissolve 10 mcg of nocodazole per 1 mL of dimethyl sulfoxide solution, making 1 tube per student. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Finally, set out all of the other tools, materials, and reagents from the list of materials onto each student&#39;s lab bench. 
</div></li></ul></li></ol>

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

Divisão Celular

Preparation of Onion Root Tips and Solutions
ExpandFor the cell cycle experiment using root tips, first, leave an onion suspended over a beaker of water ...

Processos Celulares

Physiology of the Circulatory System

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Measuring Circulatory System Function in Humans
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Before starting the experiment go to the front of the room and collect an alcohol swab, a sphygmomanometer, and a stethoscope.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Clean the earpieces with the alcohol swab and then insert them into your ears.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">To check if the stethoscope is in the on position, gently tap the flat metal disc called the diaphragm.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">If you hear a sound it&#39;s on. If not, turn the metal disc and tap again and keep turning and tapping the disc until you hear a sound. NOTE: For this experiment you will work in pairs. One person will measure their partner&#39;s blood pressure, while the person being measured will count their heartbeats over the course of one minute.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Fill out table one with the name of the person being monitored. HYPOTHESES: The experimental hypothesis is that systolic blood pressure will be higher when the person is sitting when compared to lying down because more pressure is required for blood to be moved around the body. The null hypothesis is that there will be no difference in systolic blood pressure whether a person is sitting or lying down.<a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/9/Table_1_Physiology_of_Circulatory_System.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-6"><div class="lab-step">The person being monitored should begin by lying down. Next, they will need to locate their pulse by placing two fingers next to their windpipe. If a throbbing sensation is felt, this is the pulse.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Now, ready a time keeping device to count out 1 minute as the person taking their pulse will be having their blood pressure taken at the same time.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">At this point, the person measuring blood pressure should place the cuff of the sphygmomanometer around the other arm of the subject and the stethoscope earpieces into their own ears.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Start the timer.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">The person being monitored should begin counting the number of heartbeats felt over one full minute. While the subject is taking their pulse, the person taking blood pressure should place the diaphragm of the stethoscope under the cuff on the inside of their partner&#39;s elbow.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Then, pump the cuff to 200 mm of mercury.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Gently and slowly twist the metal knob on the sphygmomanometer to release air from the cuff.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">As pressure is released the first Korotkoff sound should be heard. The corresponding value on the sphygmomanometer is the systolic blood pressure. Consistent sounds should be heard until they die out with the last sound signifying the diastolic blood pressure.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Record the systolic pressure, the diastolic pressure, and the heart rate in Table 1.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Now, repeat the blood pressure and pulse measurements with each partner keeping the same role but this time the subject should be sitting upright.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Record these data in the appropriate places in Table 1.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Finally, the partners should switch spots. So now, the person measuring blood pressure should be taking their pulse and acting as the subject.  NOTE: Be sure to wipe the earpieces of the stethoscope between users and don&#39;t forget to record your measurements in Table 1.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">After finishing the experiment, discard all alcohol swabs into the trash. Then, bring the sphygmomanometer and stethoscope back to the front of the room.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Measuring Respiratory Rate in Fish
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To prepare for this second experiment, collect two clear containers.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Begin by filling half of one container with aquarium water.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Then, use a fish net to gently catch a goldfish and place it into the cup. Pinch the net around the fish or else it may jump out of the net.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Familiarize yourself with the operculum of the fish. The operculum covers the gills and it is located behind and below the eye. When the fish respires the operculum moves in and out and this is one gill beat. HYPOTHESES: In this experiment you will measure the gill beats of the fish in different water temperatures. The hypothesis is that there will be a significant correlation between the temperature and respiration rate in goldfish. The null hypothesis is that there will be no correlation between temperature and respiration rate.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Take the other empty container with a screw cap, the cup with your fish, and the net to the water tanks.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Then, fill the empty container with the water from the lowest temperature water bath. This will act as your first respiration rate chamber.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Next, hold the fish net over a wastewater container and dump out the water from the cup holding the fish so that the fish falls into the net. Pinch the net around the fish without touching the fish itself.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Bring the net over the top of the container with the bath water and let go of the net to release the fish into the vial.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Once the fish is in the container, submerge it back into the water bath and then screw the cap on to prevent outside oxygen from entering the vial.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Next, remove the container from the controlled temperature water bath and place it on your workbench surface to avoid warming the water with body heat.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">One partner should immediately begin counting the gill beats as soon as the second partner starts the stopwatch.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Continue counting the beats for 20 seconds. Record the number of beats in column A of Table 2. <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/9/Table_2_Physiology_of_Circulatory_System.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-13"><div class="lab-step">Repeat the measurement one more time and record the number in column B.
</div></li><li data-sub-note-item="2-14"><div class="lab-step">Next, hold the fish net over a wastewater container and dump out the water from the fish vial so that the fish falls into the net.  IMPORTANT: Move from lowest to highest temperature, so the fish is not stressed between trials.
</div></li><li data-sub-note-item="2-15"><div class="lab-step">Repeat the experiment for the remaining four temperature conditions (steps 8 &#8211; 13).
</div></li><li data-sub-note-item="2-16"><div class="lab-step">Record all the values for different temperatures in Table 2.
</div></li><li data-sub-note-item="2-17"><div class="lab-step">Once you are done, slowly mix water from the last respiration chamber with the fish tank water and then return the fish to the home tank. NOTE: This should be done over the course of approximately 10 minutes.
</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">It&#39;s time to analyze the data that you just collected. Look at the blood pressure and heart rate measurements for the different trials. If they were different, make a hypothesis to explain your observations. 
</div></li><li data-sub-note-item="3-2"><div class="lab-step">For the gill beat experiment, average the numbers in columns A and B and calculate the fish respiration rate as gill beats per minute.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Next, plot the graph of temperature versus gill beats per minute.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Record any correlations you see between the variables. If a correlation is observed, determine if it is positive or negative and provide an explanation for this result.  NOTE: Consider the fact that fishes are ectotherms and the impact of temperature on dissolved oxygen.
</div></li></ul></li></ol>

Fisiologia do Sistema Circulatório

Measuring Circulatory System Function in Humans
ExpandBefore starting the experiment go to the front of the room and collect an alcohol swab, a ...

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://jove.unicartagenaproxy.elogim.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>

Isolamento de DNA e Análise de Enzimas de Restrição

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

Genética

Macromolecules

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Identifying Macromolecules with Indicator Solutions
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">HYPOTHESES: In this activity, the experimental hypothesis might be that the addition of an indicator dye to each test solution will result in a color change if the test solution contains the macromolecule of interest. The null hypothesis might be that the addition of an indicator dye will not result in a color change in any of the test solutions.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Place 16 clean test tubes in a tube rack in four rows of four tubes and label each row A through D and each column 1 - 4.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Label each test tube with its corresponding position in the test tube rack.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">After this, stir solution A thoroughly and use a pipette to add 4 mL of solution A to each tube in Row A.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Next, stir solution B until it is well mixed and use a new pipette to add 4 mL of solution B to each tube in Row B.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">After stirring solution C thoroughly, add 4 mL of solution C to each tube in Row C.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Then add 4 mL of soybean-derived vegetable oil to each tube in Row D.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">When all of the tubes have been filled with their respective test solutions, add 200 mL of water to a 400 mL beaker on a hotplate and set the hotplate to high.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">To perform the Benedict&#39;s Test, add 2 mL of Benedict&#39;s reagent to test tubes A1, B1, C1, and D1 while the water is warming.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Observe the initial color of the tubes.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">When the water begins to boil, transfer each test tube to the beaker and heat the tubes in the boiling water bath for 3 - 5 minutes.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, record the final color of each of the tubes in the appropriate cells of the table and turn off the hotplate. <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/22/Table_1_Macromolecules.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-13"><div class="lab-step">To perform an Iodine-Potassium-Iodide Test, first observe the color of the solutions in the test tubes in column 2.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Then, add 1 mL of iodine potassium iodide reagent to tubes A2 to D2 and record the final color change in each of the tubes.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">For the Sudan IV Test, observe and record the color of the solutions in the test tubes in column 3.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, add 2 - 3 drops of Sudan IV reagent to test tubes A3 through D3 and record the color change, if any, in each tube.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">For the Biuret Test, observe the color of the solutions in the test tubes in column 4.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Then, add 1 mL of Biuret reagent to test tubes A4 through D4 and record the final color in each of the tubes.
</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">Using the results from the indicator tests, each of the four original solutions can be identified as a sugar, starch, lipid, or protein. Refer to the Concepts video to help you determine which tubes contain which macromolecules.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">First, look at the results from the Benedict&#39;s Test. Determine which of the test solutions contain sugar based on your results.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Next, create a bar graph with your five plant species listed on the x-axis and the surface area in cm indicated on the y-axis.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Next, look at the results from the Iodine-Potassium-Iodide Test. Determine which of the test solutions contain starch.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Now, review the results from the Sudan IV Test. Using these observations, determine if any of the test solutions contain lipids.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Finally, look at the results from the Biuret Test. Determine if any of the test solutions contain proteins.
</div></li></ul></li></ol>

Macromolecules: Identification Using Indicator Solutions - Procedure

Macromoléculas

Identifying Macromolecules with Indicator Solutions
ExpandHYPOTHESES: In this activity, the experimental hypothesis might be that the addition of an ...