|
|
| (18 intermediate revisions by the same user not shown) |
| Line 5: |
Line 5: |
| | <link href="https://fonts.googleapis.com/css?family=Montserrat" rel="stylesheet"> | | <link href="https://fonts.googleapis.com/css?family=Montserrat" rel="stylesheet"> |
| | | | |
| − | <link href="//2015.igem.org/Team:Oxford/Assets/Bootstrap?action=raw&ctype=text/css" rel="stylesheet"> | + | |
| − | <link href="//2015.igem.org/Team:Oxford/Assets/stylesheet?action=raw&ctype=text/css" rel="stylesheet">
| + | |
| − | <link rel="stylesheet" href="//2015.igem.org/Team:Oxford/Assets/Font-Awesome?action=raw&ctype=text/css">
| + | |
| − | <script type="text/javascript" src="//2015.igem.org/Team:Oxford/Assets/Bootstrapj?action=raw&ctype=text/javascript"></script>
| + | |
| − | <script type="text/javascript" src="//2015.igem.org/Team:Oxford/Assets/contents?action=raw&ctype=text/javascript"></script>
| + | |
| − | | + | |
| − | <script src="https://2015.igem.org/common/MathJax-2.5-latest/MathJax.js?config=TeX-AMS-MML_HTMLorMML"></script>
| + | |
| | | | |
| | <link rel='stylesheet prefetch' href='https://maxcdn.bootstrapcdn.com/bootstrap/3.3.5/css/bootstrap.min.css'> | | <link rel='stylesheet prefetch' href='https://maxcdn.bootstrapcdn.com/bootstrap/3.3.5/css/bootstrap.min.css'> |
| Line 19: |
Line 13: |
| | | | |
| | <style> | | <style> |
| − | hr {
| + | @media (min-width: 768px) { |
| | + | .dropdown:hover .dropdown-menu { |
| | display: block; | | display: block; |
| − | margin-top: 5px; | + | } |
| − | margin-bottom: 15px; | + | } |
| | + | .footer{ |
| | + | display: table; |
| | + | text-align: center; |
| | margin-left: auto; | | margin-left: auto; |
| | margin-right: auto; | | margin-right: auto; |
| − |
| + | } |
| − | border-width: 1px;
| + | |
| − | | + | |
| − | } | + | |
| − | | + | |
| − | | + | |
| − | .container-fluid {
| + | |
| − | padding: 60px 50px;
| + | |
| − | }
| + | |
| − | | + | |
| − | .panel-footer .btn:hover {
| + | |
| − | border: 1px solid #f4511e;
| + | |
| − | background-color: #fff !important;
| + | |
| − | color: #f4511e;
| + | |
| − | }
| + | |
| − | | + | |
| − | .panel-footer {
| + | |
| − | background-color: white !important;
| + | |
| − | }
| + | |
| − | .panel-footer h3 {
| + | |
| − | font-size: 32px;
| + | |
| − | }
| + | |
| − | .panel-footer h4 {
| + | |
| − | color: #aaa;
| + | |
| − | font-size: 14px;
| + | |
| − | }
| + | |
| − | .panel-footer .btn {
| + | |
| − | margin: 15px 0;
| + | |
| − | background-color: #f4511e;
| + | |
| − | color: #fff;
| + | |
| − | }
| + | |
| − | | + | |
| − | footer .glyphicon {
| + | |
| − | font-size: 20px;
| + | |
| − | margin-bottom: 20px;
| + | |
| − | color: #f4511e;
| + | |
| − | }
| + | |
| − | @media screen and (max-width: 768px) {
| + | |
| − | .col-sm-4 {
| + | |
| − | text-align: center;
| + | |
| − | margin: 25px 0;
| + | |
| − | }
| + | |
| − | }
| + | |
| − | | + | |
| | body, html { | | body, html { |
| | height: 100%; | | height: 100%; |
| Line 75: |
Line 30: |
| | .main{ | | .main{ |
| | /* The image used */ | | /* The image used */ |
| − | background-image: url('#'); | + | background-image: url('https://static.igem.org/mediawiki/2017/e/eb/IMG_0041.jpg'); |
| | | | |
| | /* Full height */ | | /* Full height */ |
| Line 87: |
Line 42: |
| | opacity: 0.9; | | opacity: 0.9; |
| | | | |
| | + | } |
| | + | .centered { |
| | + | position: absolute; |
| | + | top: 200px; |
| | + | left: 250px; |
| | + | |
| | + | color: white; |
| | } | | } |
| | | | |
| Line 177: |
Line 139: |
| | padding: 0; | | padding: 0; |
| | background-color: white; | | background-color: white; |
| | + | margin-top: 9px; |
| | /* Add additional styles here for the COLLAPSED state */ | | /* Add additional styles here for the COLLAPSED state */ |
| | } | | } |
| Line 455: |
Line 418: |
| | <a class="dropdown-toggle" data-toggle="dropdown" href="#">PROJECT <span class="caret"> </span></a> | | <a class="dropdown-toggle" data-toggle="dropdown" href="#">PROJECT <span class="caret"> </span></a> |
| | <ul class="dropdown-menu"> | | <ul class="dropdown-menu"> |
| − | <li><a href="https://2017.igem.org/Team:CMUQ/ descritpion">Description</a></li> | + | <li><a href="https://2017.igem.org/Team:CMUQ/ Description">Description</a></li> |
| − | <li><a href="https://2017.igem.org/Team:CMUQ/design">Design</a></li> | + | <li><a href="https://2017.igem.org/Team:CMUQ/Design">Design</a></li> |
| − | <li><a href="https://2017.igem.org/Team:CMUQ/safety">Safety</a></li> | + | <li><a href="https://2017.igem.org/Team:CMUQ/Safety">Safety</a></li> |
| | <li><a href="https://2017.igem.org/Team:CMUQ/Demonstrate">Demonstrate</a></li> | | <li><a href="https://2017.igem.org/Team:CMUQ/Demonstrate">Demonstrate</a></li> |
| − | <li><a href="https://2017.igem.org/Team:CMUQ/Model">Model</a></li> | + | <li><a href="https://2017.igem.org/Team:CMUQ/Parts">Parts</a></li> |
| | <li><a href="https://2017.igem.org/Team:CMUQ/Collaborations">Collaborations</a></li> | | <li><a href="https://2017.igem.org/Team:CMUQ/Collaborations">Collaborations</a></li> |
| − | <li><a href="https://2017.igem.org/Team:CMUQ/achievments">Achievements</a></li> | + | <li><a href="https://2017.igem.org/Team:CMUQ/JudgingForm">Judging Form </a></li> |
| | + | |
| | </ul> | | </ul> |
| | </li> | | </li> |
| | <li class="dropdown"> | | <li class="dropdown"> |
| − | <a class="dropdown-toggle" data-toggle="dropdown" href="#"> PRACTICES <span class="caret"> </span></a> | + | <a class="dropdown-toggle" data-toggle="dropdown" href="#"> COMMUNITY INVOLVEMENT <span class="caret"> </span></a> |
| | <ul class="dropdown-menu"> | | <ul class="dropdown-menu"> |
| | <li><a href="https://2017.igem.org/Team:CMUQ/humanpractices">Human Practices</a></li> | | <li><a href="https://2017.igem.org/Team:CMUQ/humanpractices">Human Practices</a></li> |
| | <li><a href="https://2017.igem.org/Team:CMUQ/HP/Silver"> Silver HP </a></li> | | <li><a href="https://2017.igem.org/Team:CMUQ/HP/Silver"> Silver HP </a></li> |
| | + | <li><a href="https://2017.igem.org/Team:CMUQ/HP/Gold_Integrated"> Gold HP </a></li> |
| | + | <li><a href="https://2017.igem.org/Team:CMUQ/Engagement"> Outreach</a></li> |
| | </ul> | | </ul> |
| | </li> | | </li> |
| | <li class="dropdown"> | | <li class="dropdown"> |
| − | <a class="dropdown-toggle" data-toggle="dropdown" href="#"> WET LAB <span class="caret"> </span></a> | + | <a class="dropdown-toggle" data-toggle="dropdown" href="#"> LAB WORK <span class="caret"> </span></a> |
| | <ul class="dropdown-menu"> | | <ul class="dropdown-menu"> |
| − | <li><a href="https://2017.igem.org/Team:CMUQ/results">Overview & Results</a></li> | + | |
| − | <li><a href="https://2017.igem.org/Team:CMUQ/notebook">Notebook</a></li> | + | <li><a href="https://2017.igem.org/Team:CMUQ/Notebook">Notebook</a></li> |
| − | <li><a href="https://2017.igem.org/Team:CMUQ/protocols">Protocols</a></li> | + | <li><a href="https://2017.igem.org/Team:CMUQ/protocols">Procedures</a></li> |
| − | <li><a href="https://2017.igem.org/Team:CMUQ/InterLab">InterLab</a></li> | + | <li><a href="https://2017.igem.org/Team:CMUQ/InterLab">InterLab Study</a></li> |
| − | </ul>
| + | <li><a href="https://2017.igem.org/Team:CMUQ/ Results"> Results</a></li> |
| − | </li>
| + | |
| − | <li class="dropdown">
| + | |
| − | <a class="dropdown-toggle" data-toggle="dropdown" href="#">DRY LAB<span class="caret"> </span></a>
| + | |
| − | <ul class="dropdown-menu">
| + | |
| − | <li><a href="https://2017.igem.org/Team:CMUQ/ modelling"> Modelling</a></li> | + | |
| | </ul> | | </ul> |
| | </li> | | </li> |
| | + | |
| | <li class="dropdown"> | | <li class="dropdown"> |
| | <a class="dropdown-toggle" data-toggle="dropdown" href="#"> TEAM <span class="caret"> </span></a> | | <a class="dropdown-toggle" data-toggle="dropdown" href="#"> TEAM <span class="caret"> </span></a> |
| | <ul class="dropdown-menu"> | | <ul class="dropdown-menu"> |
| − | <li><a href="https://2017.igem.org/Team:CMUQ/team">Team</a></li> | + | <li><a href="https://2017.igem.org/Team:CMUQ/Team">Team</a></li> |
| − | <li><a href="https://2017.igem.org/Team:CMUQ/attributions">Attributions</a></li> | + | <li><a href="https://2017.igem.org/Team:CMUQ/Attributions">Attributions</a></li> |
| | </ul> | | </ul> |
| | </li> | | </li> |
| Line 503: |
Line 465: |
| | | | |
| | <div class="main"> | | <div class="main"> |
| − | | + | <div class="centered"> <h1>Protocols </h1> </div> |
| | </div> | | </div> |
| | | | |
| | <body> | | <body> |
| | | | |
| | + | <div> |
| | + | <button type="button" class="btn btn-default btn-lg btn-block" style= "font-size: 35px;"><a href="https://2017.igem.org/Team:CMUQ/BioSensor_Lab"> BioSensor Lab<a/></button> |
| | + | <button type="button" class="btn btn-default btn-lg btn-block" style= "font-size: 35px;"><a href="https://2017.igem.org/Team:CMUQ/Dspb_Lab">DspB Lab<a/></button> |
| | + | <button type="button" class="btn btn-default btn-lg btn-block" style= "font-size: 35px;"><a href="https://2017.igem.org/Team:CMUQ/Biofilm_Formation_Lab">Biofilm Formation Lab<a/></button> |
| | + | <button type="button" class="btn btn-default btn-lg btn-block" style= "font-size: 35px;"><a href="https://2017.igem.org/Team:CMUQ/InterLab">Interlab<a/></button> |
| | + | |
| | + | |
| | + | |
| | + | </div> |
| | | | |
| − | <div class = "division" style = "padding: 35px;"> | + | <div class="footer"> |
| − | <p style = "font-size: 60px;"> BioSensor Lab </p>
| + | |
| − | <h3>A1: T7 salt sensor </h3>
| + | |
| − | <hr>
| + | |
| − | <h4> Introduction </h4>
| + | |
| − | <p> The goal is to amplify T7 salt sensor, digest it along with pSB1C3 plasmid with EcoRI and PstI</p>
| + | |
| − | <h4>Materials </h4>
| + | |
| − | <ul>
| + | |
| − | <li> Polymerase: Fusion Taq or Ampli tas (gold) </li>
| + | |
| − | <li>Primers: PrefixF and SuffixR </li>
| + | |
| − | <li>Nuclease free H2O </li>
| + | |
| − | <li>5xHF PCR Buffer </li>
| + | |
| − | <li>gBlock DNA </li>
| + | |
| − | <li> dNTPs</li>
| + | |
| − | <li>PCR tubes </li>
| + | |
| − | </ul>
| + | |
| − | | + | |
| − | <h4> Procedure </h4>
| + | |
| − | <p style= "font-size: 15px; padding: 15px;"> Preparation of reagents </p>
| + | |
| − | | + | |
| − | <ol>
| + | |
| − | <li> Resuspend gBLOCK in 20µL nuclease free dH2O</li>
| + | |
| − | <li> Resuspend primers to make 100µM stock then dilute to make a 10µM stock</li>
| + | |
| − | <li>Add the following reagents to a PCR tube: </li>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/e/ef/Table_a1.png" width = 30% position="center">
| + | |
| − | <li>Run the PCR machine </li>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/2/21/A1_table2.png" width = 50% position="center">
| + | |
| − | <li> Digest amplified sequence with EcoRI and PstI </li>
| + | |
| − | <li> Digest pSB1C3 vector with EcoRI and PstI</li>
| + | |
| − | <li> If you clone into a vector that expresses a fluorescent protein, other than red since you are cloning in red, then colonies that still express that fluorescent protein will be negative clones and the red or white colonies will be the ones that you want.</li>
| + | |
| − | <li>Run on an agarose gel, cut out the bands and purify. </li>
| + | |
| − | <li> Send clones for sequencing </li>
| + | |
| − | </ol>
| + | |
| − | | + | |
| − | | + | |
| − | <h3>A2: PCR Purification </h3>
| + | |
| − | <hr>
| + | |
| − | <h4> Introduction </h4>
| + | |
| − | <p> Adapted from: <a href="https://www.qiagen.com/us/resources/download.aspx?id=390a728a-e6fc-43f7-bf59-b12091cc4380&lang=en">Here </a> </p>
| + | |
| − | <h4>Materials </h4>
| + | |
| − | <p>The QIAquick PCR Purification Kit (cat. nos. 28104 and 28106) can be stored at room temperature (15–25°C) for up to 12 months. </p>
| + | |
| − | | + | |
| − | <h4> Procedure </h4>
| + | |
| − | <h4 style= "padding: 10px;">Notes before starting: </h4>
| + | |
| − | <ol>
| + | |
| − | <li> Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).</li>
| + | |
| − | | + | |
| − | <li> All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge at room temperature.</li>
| + | |
| − | <li>Add 1:250 volume pH indicator I to Buffer PB. The yellow color of Buffer PB with pH indicator I indicates a pH of ≤7.5. If the purified PCR product is to be used in sensitive microarray applications, it may be beneficial to use Buffer PB without the addition of pH indicator I. Do not add pH indicator I to buffer aliquots. </li>
| + | |
| − | </ol>
| + | |
| − | <h4> Protocol </h4>
| + | |
| − | <ol>
| + | |
| − | | + | |
| − | <li>Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.</li>
| + | |
| − | | + | |
| − | <li>Place a QIAquick column in a provided 2 ml collection tube or into a vacuum manifold. For details on how to set up a vacuum manifold, refer to the QIAquick Spin Handbook.</li>
| + | |
| − | <li> To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s or apply vacuum to the manifold until all the samples have passed through the column. Discard flow-through and place the QIAquick column back in the same tube.</li>
| + | |
| − | | + | |
| − | <li> To wash, add 0.75 ml Buffer PE to the QIAquick column centrifuge for 30–60 s or apply vacuum. Discard flow-through and place the QIAquick column back in the same tube.</li>
| + | |
| − | <li>Centrifuge the QIAquick column once more in the provided 2 ml collection tube for 1 min to remove residual wash buffer. </li>
| + | |
| − | <li> Place each QIAquick column in a clean 1.5 ml microcentrifuge tube. </li>
| + | |
| − | <li>To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0– 8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge. </li>
| + | |
| − | <li>If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel. </li>
| + | |
| − | </ol>
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h3>A3: Gel Extraction </h3>
| + | |
| − | <hr>
| + | |
| − | <h4> Introduction </h4>
| + | |
| − | <p> Adapted from: <a href="https://www.qiagen.com/us/resources/download.aspx?id=8a1e1b5f-70be-480b-bc9d-8eb444ff7b63&lang=en">Here</a> </p>
| + | |
| − | <h4>Materials </h4>
| + | |
| − | <p>The QIAEX II Gel Extraction Kit (cat. nos. 20021 and 20051) can be stored at
| + | |
| − | room temperature (15–25°C) for up to 12 months. </p>
| + | |
| − | | + | |
| − | <h4> Procedure </h4>
| + | |
| − | | + | |
| − | <h4 style= "padding: 10px;">Notes before starting: </h4>
| + | |
| − | <ol>
| + | |
| − | <li> This protocol is for cleanup of DNA fragments of 40 bp to 50 kb. The yellow color of Buffer QX1 indicates a pH ≤7.5.</li>
| + | |
| − | | + | |
| − | <li> Add ethanol (96–100%) to Buffer PE concentrate before use (see bottle
| + | |
| − | label for volume).</li>
| + | |
| − | <li> A heating block or water bath at 50°C is required.</li>
| + | |
| − | <li>All centrifugation steps are carried out at 17,900 x g (~13,000 rpm) in a conventional tabletop microcentrifuge at room temperature (15–25°C). </li>
| + | |
| − | <li> For purification of DNA from polyacrylamide gels or aqueous solutions, see the handbook. </li>
| + | |
| − | </ol>
| + | |
| − | <h4> Protocol </h4>
| + | |
| − | <ol>
| + | |
| − | | + | |
| − | <li>Excise the DNA band from the agarose gel with a clean, sharp scalpel. Use a 1.5 ml microfuge tube for processing up to 250 mg agarose per tube.</li>
| + | |
| − | | + | |
| − | <li>Weigh the gel slice in a colorless tube. Add Buffer QX1 according to DNA fragment size: 6 volumes for <100 bp; 3 volumes for 100 bp – 4 kb; 3 volumes with 2 volumes of water for >4 kb. Add 6 volumes of Buffer QX1 when using >2% or Metaphor agarose gels.</li>
| + | |
| − | | + | |
| − | <li> Resuspend QIAEX II by vortexing for 30 s. Add QIAEX II to the sample and mix: Use 10 μl QIAEXII for ≤2 μg DNA; 30 μl for 2–10 μg DNA; and an additional 30 μl for each additional 10 μg DNA.</li>
| + | |
| − | | + | |
| − | <li> Incubate at 50°C for 10 min to solubilize the agarose and bind the DNA. Mix by vortexing* every 2 min to keep QIAEX II in suspension. Check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The color should turn to yellow. The incubation should then be continued for at least 5 min.</li>
| + | |
| − | | + | |
| − | <li>Centrifuge the sample for 30 s and carefully remove supernatant with a pipet. </li>
| + | |
| − | | + | |
| − | <li> Wash the pellet with 500 μl Buffer QX1. Resuspend the pellet by vortexing.* Centrifuge the sample for 30 s and remove all traces of supernatant with a pipet. This wash step removes residual agarose contaminants.</li>
| + | |
| − | | + | |
| − | <li>Wash the pellet twice with 500 μl Buffer PE. Resuspend the pellet by vortexing.* Centrifuge the sample for 30s and carefully remove all traces of supernatant with a pipet. This step removes residual salt contaminants.</li>
| + | |
| − | <li>Air-dry the pellet for 10–15 min or until the pellet becomes white. If 30 μl QIAEX II suspension is used, air-dry the pellet for approximately 30 min. Do not vacuum dry, as overdrying, may lead to decreased elution efficiency.</li>
| + | |
| − | <li>To elute DNA, add 20 μl of 10 mM Tris·Cl, pH 8.5, TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), or water and resuspend the pellet by vortexing.* Incubate according to the DNA fragment size: 5 min at room
| + | |
| − | temperature (15–25°C) for ≤4 kb; 5 min at 50°C for 4–10 kb; or 10 min at 50°C for >10 kb. </li>
| + | |
| − | <li>Centrifuge for 30 s, and carefully pipet the supernatant into a clean tube. The supernatant now contains the purified DNA. </li>
| + | |
| − | <li>Optional: repeat steps 9 and 10 and combine the eluates. A second
| + | |
| − | elution step will increase the yield by approximately 10–15%. </li>
| + | |
| − | | + | |
| − | <p> For fragments larger than 10 kb, resuspend the pellet by inverting and flicking the tube. Vortexing can
| + | |
| − | cause shearing of large DNA fragments. </p>
| + | |
| − | | + | |
| − | </ol>
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <p style = "font-size: 60px;"> DspB Lab </p>
| + | |
| − | <h3>B1: Agarose gel </h3>
| + | |
| − | <hr>
| + | |
| − | | + | |
| − | <h4>Materials </h4>
| + | |
| − | <ul>
| + | |
| − | <li> Agarose</li>
| + | |
| − | <li>dH2O </li>
| + | |
| − | <li>Ethedium Bromide </li>
| + | |
| − | <li>10X TBE </li>
| + | |
| − | <li>Gel box + power </li>
| + | |
| − | <li> DNA ladder (check size)</li>
| + | |
| − | <li>DNA loading dye </li>
| + | |
| − | </ul>
| + | |
| − | | + | |
| − | <h4> Procedure </h4>
| + | |
| − | <p style= "font-size: 15px; padding: 15px;">Making the gel: </p>
| + | |
| − | | + | |
| − | <ol>
| + | |
| − | <li> Weigh appropriate mass of Agarose into an Erlenmeyer flask</li>
| + | |
| − | <li> Add appropriate volume of dH2O and 10% TBE, For example, to make 1% mini gel: 0.5 g agarose + 45 mL dH2O + 5 mL 10x TBE</li>
| + | |
| − | <li>Microwave for 2 min, take out every 30 sec and swirl to dissolve </li>
| + | |
| − | | + | |
| − | | + | |
| − | <li>Using hot hand protectors, take to bench and let cool </li>
| + | |
| − | | + | |
| − | <li>Add appropriate volume of Ethidium Bromide and swirl to mix</li>
| + | |
| − | | + | |
| − | <li> Pour onto previously setup gel box with comb</li>
| + | |
| − | <li> Allow to solidify for ≈20 min</li>
| + | |
| − | <li>Add 1% TBE </li>
| + | |
| − | <li> Run for 30 - 45 min at 100 Volts </li>
| + | |
| − | <li> Image and discard gel</li>
| + | |
| − | </ol>
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h3>B2: LB Media Preparation</h3>
| + | |
| − | <hr>
| + | |
| − | | + | |
| − | <h4>Materials </h4>
| + | |
| − | <ul>
| + | |
| − | <li> Tryptone</li>
| + | |
| − | <li> NaCl </li>
| + | |
| − | <li>Yeast extract </li>
| + | |
| − | <li>dH2O </li>
| + | |
| − | <li>Autoclave</li>
| + | |
| − | <li> 1N NaOH</li>
| + | |
| − | | + | |
| − | </ul>
| + | |
| − | | + | |
| − | <h4> Procedure </h4>
| + | |
| − | | + | |
| − | | + | |
| − | <ol>
| + | |
| − | <li> Dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 950 mL deionized water.</li>
| + | |
| − | <li> Adjust the pH of the medium to 7.0 using 1N NaOH and bring volume up to 1 liter.</li>
| + | |
| − | <li>Autoclave on liquid cycle for 20 min at 15 psi. Allow solution to cool to 60°C. </li>
| + | |
| − | | + | |
| − | | + | |
| − | <li>Store in the fridge. </li>
| + | |
| − | | + | |
| − | | + | |
| − | </ol>
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <h3>B3: SOC Media Preparation</h3>
| + | |
| − | <hr>
| + | |
| − | | + | |
| − | <h4>Introduction </h4>
| + | |
| − | <p>Protocol adapted from: <a hred="http://aix1.uottawa.ca/~sgee/protocols/socmedium.htm">Here </a> </p>
| + | |
| − | <ul>
| + | |
| − | <li> Tryptone</li>
| + | |
| − | <li> Yeast Extract </li>
| + | |
| − | <li>NaCl </li>
| + | |
| − | <li>250 mM KCL </li>
| + | |
| − | <li> 1N NaOH</li>
| + | |
| − | <li> 1M glucose</li>
| + | |
| − | <li>2M MgCl2</li>
| + | |
| − | <li>dH2O</li>
| + | |
| − | | + | |
| − | </ul>
| + | |
| − | | + | |
| − | | + | |
| − | <h3>B4: LB Chl Plates Prep</h3>
| + | |
| − | <hr>
| + | |
| − | <h4> Introduction </h4>
| + | |
| − | <p> Adapted from: <a href="https://www.addgene.org/protocols/pouring-lb-agar-plates/
| + | |
| − | https://www.addgene.org/mol-bio-reference/antibiotics/">Here</a> </p>
| + | |
| − | <p>Genetics lab recipe book in 2033 </p>
| + | |
| − | | + | |
| − | <h4>Materials </h4>
| + | |
| − | <ul>
| + | |
| − | <li>Antibiotic: Chloramphenicol </li>
| + | |
| − | <li>De-ionized Water </li>
| + | |
| − | <li>LB-Agar </li>
| + | |
| − | <li> Plates with lids</li>
| + | |
| − | <li>Erlenmeyer flasks </li>
| + | |
| − | <li>60˚C water bath </li>
| + | |
| − | <li> Autoclave</li>
| + | |
| − | <li>Flame </li>
| + | |
| − | </ul>
| + | |
| − | | + | |
| − | <h4> Procedure </h4>
| + | |
| − | | + | |
| − | <h4 >Preparing 250 mL LB-Agar </h4>
| + | |
| − | <ol>
| + | |
| − | <li> Add the following into a 500 mL Erlenmeyer flask:</li>
| + | |
| − | <img src= "https://static.igem.org/mediawiki/2017/2/27/B4_table1.png" width= 40%>
| + | |
| − | | + | |
| − | <li> Swirl flask and cover the opening with aluminum foil and tape the bottle with autoclave tape. </li>
| + | |
| − | | + | |
| − | <li> Label the bottle with your initials, the date, and the bottle contents.</li>
| + | |
| − | <li>Place the gel mix in the autoclave and run on a setting that gets the sample to at least 121 ℃ under 20 psi for at least 30 min. The high pressure will prevent your gel mix from boiling over at high temperature.</li>
| + | |
| − | | + | |
| − | <li> Turn the water bath and heat to 60˚C</li>
| + | |
| − | <li>After 1 - 1.5 hours the LB agar autoclave was complete, the flask was taken using rubber hot hand protector and placed in a 60˚C water bath </li>
| + | |
| − | | + | |
| − | <h4>Preparing Chloramphenicol antibiotic </h4>
| + | |
| − | | + | |
| − | <li>Brenadette weighed 12.5 mg chroamphinicol in powder form (box in the 4˚C fridge) into a 2.5 mL microcentrifuge tube</li>
| + | |
| − | | + | |
| − | <li>Dissolve in 500 µL ethanol to make 25mg/mL (I used 70% Ethanol)</li>
| + | |
| − | | + | |
| − | <li> Vortex to mix and leave on a rack untill 10 min has elapsed on the LB agar solution in water bath and the pouring station is prepared</li>
| + | |
| − | | + | |
| − | <h4>Pour Plating</h4>
| + | |
| − | | + | |
| − | <li>Use the hood next to the 4˚C fridges in 2033</li>
| + | |
| − | | + | |
| − | <li>Spray down the bench with bleach and 70% ethanol solution and wipe down with a paper towel.</li>
| + | |
| − | | + | |
| − | <li> Obtain a stack (20) small plates, and stack them in the hood.</li>
| + | |
| − | | + | |
| − | <li>Open the plates and place the lids on the side</li>
| + | |
| − | <li>Obtain LB Agar flask from water bath, add 250 µL of 25 mg/mL Chloramphenicol and swirl to mix</li>
| + | |
| − | <li>Pour directly from the Erlenmeyer flask, a little more than half of the plate.</li>
| + | |
| − | <li>Once all the plates are poured, ignite the flame and quickly run over the plates to avoid contamination. </li>
| + | |
| − | <li>Leave your plates to solidify. it takes ≈ 20 min to solidify at room temperature. </li>
| + | |
| − | <li> Close the plates with the lids. and stack on top of each other.</li>
| + | |
| − | <li>Label the plates withblue and orange markers coding for Chloramphenicol LB </li>
| + | |
| − | <li>Label the plates with the date and initials </li>
| + | |
| − | <li> The plates are then placed in a box with iGEM label and stored in 4 ℃ fridge.</li>
| + | |
| − | | + | |
| − | | + | |
| − | </ol>
| + | |
| | | | |
| | + | <span class="glyphicon glyphicon-chevron-up"></span> |
| | | | |
| | + | </br> |
| | + | <img src = "https://static.igem.org/mediawiki/2017/e/e5/Occidental_Logo..png" width = "15%;" > |
| | + | <img src = "https://static.igem.org/mediawiki/2017/6/66/Logo_w-o_bg_green_mangroves.png" width = "20%;" > |
| | + | <img src = "https://static.igem.org/mediawiki/2017/f/fa/IDT_logo.cmuq.png" width = "20%;" > |
| | + | <img src = "https://static.igem.org/mediawiki/2017/5/53/Benchling-logo.png" width = "20%; " > |
| | </div> | | </div> |
| − |
| |
| − |
| |
| − |
| |
| | | | |
| | | | |
