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| - | <style type="text/css">
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| - | #globalWrapper {background-color: #153E7E;}
| + | <h1>Click on the happy scientist to open the protocol |
| - | #top-section { height: 14px; margin: 0px; margin-left: auto; margin-right: auto; margin-bottom: 0 !important;}
| + | </h1> |
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| + | <Br> |
| - | #menubar.left-menu { background-color:#606060; width:965px; float:left; border: none;}
| + | High Efficiency Transformation Protocol |
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| + | <br> |
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| + | [[File:Happy_scientist.gif|High Efficiency Transformation Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/transformation]] |
| - | #contentgrid {padding-left:25px;padding-right:25px;width:925px;border: 1px solid #529bc7background-color: white; margin-left:-6px; margin-bottom:-5px; }
| + | <br> |
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| + | Culture Preparation Protocol |
| - | | + | <br> |
| - | #headerimage {width:965px; margin-bottom:10px}
| + | [[File:Happy_scientist.gif|Culture Preparation Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/cultureprep]] |
| - | #headerimage img {max-width:965px;}
| + | <br> |
| - | #headermenu {text-align:center; width:965px; height:22px;border-bottom:1px solid #529bc7;}
| + | Glycerol Stock Solution Protocol |
| - | #headermenu ul {display:inline-block; zoom:1;}
| + | <br> |
| - | #headermenu ul.top {margin-left:0;}
| + | [[File:Happy_scientist.gif|Glycerol Stock Solution Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/glycerolstock]] |
| - | #headermenu ul {text-align:left; margin:0; padding:0; list-style:none; white-space:nowrap;}
| + | <br> |
| - | #headermenu li {margin:0; padding:0;}
| + | Qiagen Miniprep Protocol |
| - | #headermenu a {display:block; font:normal 11px verdana,arial,sans-serif;color:#82CAFA; line-height:22px; text-decoration:none; padding:0 20px;}
| + | <br> |
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| + | [[File:Happy_scientist.gif|Qiagen Miniprep Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/miniprep]] |
| - | #headermenu a:hover ul,
| + | <br> |
| - | #headermenu a:hover a:hover ul,
| + | Gel Electrophoresis Protocol |
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| + | <br> |
| - | #headermenu a:hover ul ul,
| + | [[File:Happy_scientist.gif|Gel Electrophoresis Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/electrophoresis]] |
| - | #headermenu a:hover a:hover ul ul {visibility:hidden;}
| + | <br> |
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| + | PCR Protocol |
| - | #headermenu li.top-li {float:left; position:relative; margin-right:1px;}
| + | <br> |
| - | #headermenu a.top-a {float:left; padding:0 0 0 20px; background:url(https://static.igem.org/mediawiki/2011/2/2f/Screen_shot_2011-06-23_at_14.05.42.png) no-repeat left top;}
| + | [[File:Happy_scientist.gif|PCR Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/pcr]] |
| - | #headermenu a.top-a b {float:left; padding:0 20px 0 0; background:url(https://static.igem.org/mediawiki/2011/2/22/Screen_shot_2011-06-23_at_14.01.36.png) no-repeat right top; cursor:pointer; cursor:hand;}
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| + | PEG Transformation Protocol |
| - | #headermenu a.top-a:hover {white-space:nowrap; background:url(https://static.igem.org/mediawiki/2011/2/2f/Screen_shot_2011-06-23_at_14.05.42.png) no-repeat left -30px;}
| + | <br> |
| - | #headermenu a.top-a:hover b,
| + | [[File:Happy_scientist.gif|PEG Transformation Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/pegtransformation]] |
| - | #headermenu a.top-a:focus b,
| + | <br> |
| - | #headermenu a.top-a:active b {color:#FDD017; background:url(https://static.igem.org/mediawiki/2011/3/33/Tab_new_1.png) no-repeat right -30px; cursor:pointer;}
| + | Algae Transformation using glass beads |
| - | #headermenu a.down:hover b,
| + | <br> |
| - | #headermenu a.down:focus b,
| + | [[File:Happy_scientist.gif|Algae Transformation using glass beads Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/algaeglassbeads]] |
| - | #headermenu a.down:active b {color:#000; background:url(https://static.igem.org/mediawiki/2011/3/33/Tab_new_1.png) no-repeat right -30px; cursor:pointer;}
| + | <br> |
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| + | Algae Transformation using electroporation |
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| + | <br> |
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| + | [[File:Happy_scientist.gif|Algae Transformation using electroporation Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/algaeelectroporation]] |
| - | #headermenu li ul {display:block; position:absolute; visibility:hidden; background:#529bc7; padding:1px 1px 8px 1px; left:0;}
| + | <br> |
| - | #headermenu li li {border-bottom:1px solid #529bc7;}
| + | Site Directed Mutagenesis Protocol |
| - | #headermenu li li a {background:#fff;}
| + | <br> |
| - | #headermenu li li a:hover {background:#153E7E;}
| + | [[File:Happy_scientist.gif|Site Directed Mutagenesis Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/sdm]] |
| - | #headermenu li li:hover > a {background:#153e7e;}
| + | <br> |
| - | #headermenu ul.drop-down {top:22px; opacity:1.0;}
| + | pGEM T-easy Vector Cloning protocol |
| - | #headermenu li li ul {left:100%; margin-top:-23px; margin-left:-5px;}
| + | <br> |
| - | #headermenu table {text-align:left; position:absolute;top:0;left:0;border-collapse:collapse;}
| + | [[File:Happy_scientist.gif|pGEM T-easy Vector Cloning Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/vectorcloning]] |
| - | #headermenu table ul li a {padding-left:0; padd\ing-left:20px;}
| + | <br> |
| - | #headermenu table table {top:auto; left:100%; margin-left:-1px; padding:0; margin:0;}
| + | Restriction Digest protocol |
| - | #headermenu table table ul {margin-top:-4px; marg\in-top:-7px;}
| + | <br> |
| - | </style> | + | [[File:Happy_scientist.gif|Restriction Digest Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/rdigest]] |
| - | <div id="wrap"> | + | <br> |
| - | <div id="headermenu"> | + | Poly "A" Tailing Protocol |
| - | <ul class="top">
| + | <br> |
| - | <li class="top-li"><a class="top-a" href="https://2011.igem.org/Team:UEA-JIC_Norwich"><b>Home</b></a>
| + | [[File:Happy_scientist.gif|Poly "A" Tailing PCR Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/polyatailing]] |
| - | </li> | + | <br> |
| - |
| + | Ligation Protocol |
| - | <li class="top-li"><a class="top-a" href="https://2011.igem.org/Team:UEA-JIC_Norwich/Team"><b>Team</b></a>
| + | <br> |
| - |
| + | [[File:Happy_scientist.gif|Ligation Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/ligation]] |
| - | </li>
| + | <br> |
| - |
| + | Gel Extraction Protocol |
| - | <li class="top-li"><a class="top-a down" href=""><b>Aims</b>
| + | <br> |
| - | <ul class="drop-down">
| + | [[File:Happy_scientist.gif|Gel Extraction Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/gelextract]] |
| - | <li><a href="https://2011.igem.org/Team:UEA-JIC_Norwich/Project">Overview</a></li>
| + | |
| - | <li><a href="https://2011.igem.org/Team:UEA-JIC_Norwich/Nittygritty-bacteria">Bacteria</a></li>
| + | |
| - | <li><a href="https://2011.igem.org/Team:UEA-JIC_Norwich/Nittygritty-algae">Algae</a></li>
| + | |
| - | <li><a href="https://2011.igem.org/Team:UEA-JIC_Norwich/Nittygritty-moss">Moss</a></li>
| + | |
| - | </ul>
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| - | </li>
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| - |
| + | |
| - | <li class="top-li"><a class="top-a down" href=""><b>Registry Parts</b>
| + | |
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| + | |
| - | <li><a href="https://2011.igem.org/Team:UEA-JIC_Norwich/Registryoverview">Overview</a></li>
| + | |
| - | <li><a href="https://2011.igem.org/Team:UEA-JIC_Norwich/Registrycharacterisation">Characterization</a></li>
| + | |
| - | </ul>
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| - | </li>
| + | |
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| - | <li class="top-li"><a class="top-a down" href="https://2011.igem.org/Team:UEA-JIC_Norwich/Methods"><b>Methods</b>
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| - | </li>
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| - | <li class="top-li"><a class="top-a down" href=""><b>Journal</b>
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| - | <li><a href="https://2011.igem.org/Team:UEA-JIC_Norwich/Notebook">Lab Journal</a></li>
| + | |
| - | <li><a href="https://2011.igem.org/Team:UEA-JIC_Norwich/DaytoDay">Day to Day Journal</a></li>
| + | |
| - | </ul>
| + | |
| - | </li>
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| - |
| + | |
| - | | + | |
| - | <li class="top-li"><a class="top-a" href="https://2011.igem.org/Team:UEA-JIC_Norwich/Safety"><b>Lab Safety</b></a>
| + | |
| - | </li> | + | |
| - |
| + | |
| - | <li class="top-li"><a class="top-a down" href=""><b>Human Practices</b></a>
| + | |
| - | <ul class="drop-down"> | + | |
| - | <li><a href="top-a down" href="https://2011.igem.org/Team:UEA-JIC_Norwich/Outreach">Outreach</a></li>
| + | |
| - | <li><a href="https://2011.igem.org/Team:UEA-JIC_Norwich/Energyconservation">Energy Conservation</a></li>
| + | |
| - | </ul></li>
| + | |
| - | <li class="top-li"><a class="top-a" href="https://2011.igem.org/Team:UEA-JIC_Norwich/Media"><b>Media</b></a>
| + | |
| - | </li> | + | |
| - | <li class="top-li"><a class="top-a" href="https://2011.igem.org/Team:UEA-JIC_Norwich/Follow_us"><b>Follow Us</b></a>
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| - | <h2>High Efficiency Transformation Protocol</h2>
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| - | <p>1. Thaw a tube of NEB 5-alpha Competent E.coli cells on ice for 10 minutes</p> | + | |
| - | <p>2. Mark the location of the Biobrick well (letters from top to bottom, numbers from left to right)</p>
| + | |
| - | <p>3. Resuspend specific biobrick part (1 μl) with distilled water (20 μl). Aspirate up and down a few times</p>
| + | |
| - | <p>4. Inoculate with DNA ( 1 μl) to the competent cells</p>
| + | |
| - | <p>5. Place the mixture on ice for 30 minutes. Do not mix</p>
| + | |
| - | <p>6. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix</p>
| + | |
| - | <p>7. Place on ice for 5 minutes. Do not mix</p>
| + | |
| - | <p>8. Pipette 950 μl of room temperature SOC into the mixture</p>
| + | |
| - | <p>9. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate</p>
| + | |
| - | <p>10. Warm selection plates to 37°C</p>
| + | |
| - | <p>11. Do serial dilutions (2-3) at 105. Mix cells, flick/invert gently</p>
| + | |
| - | <p>12. Spread (100 μl) on agar plates of each dilution</p>
| + | |
| - | <p>13. Incubate overnight at 37°C</p>
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| - | | + | |
| - | <h2>Culture Preparation Protocol</h2>
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| - | | + | |
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| - | <p>1. Select a colony by sampling it with a wooden pick</p>
| + | |
| - | <p>2. Place into the appropriate antibiotic LB broth</p>
| + | |
| - | <p>3. Incubate at 37°C overnight in an Incubator Shaker</p>
| + | |
| - | <p>4. Store at -20°C</p>
| + | |
| - | | + | |
| - | | + | |
| - | <h2>Glycerol Stock Solution Protocol</h2>
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| - | | + | |
| - | | + | |
| - | <p>1. Take 500µL of a previously prepared culture and place into a screw-top eppendorf tube</p>
| + | |
| - | <p>2. Add 500µL of Glycerol solution (V.V 50% to make a 25% concentration</p>
| + | |
| - | <p>3. Place in -20°C storage</p>
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| - | <h2>Qiagen miniprep Protocol</h2>
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| - | <p>1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube. </p>
| + | |
| - | <p>2. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, solution turns blue. </p>
| + | |
| - | <p>3. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, solution turns colorless. </p>
| + | |
| - | <p>4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. </p>
| + | |
| - | <p>5. Apply the supernatant (from step 4) to the QIAprep spin column by decanting or pipetting. </p>
| + | |
| - | <p>6. Centrifuge for 30–60 s. Discard the flow-through. </p>
| + | |
| - | <p>7. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through. This step is only required when using endA+ or other bacteria strains with high nuclease activity or carbohydrate content (see QIAprep Miniprep Handbookfor more details) </p>
| + | |
| - | <p>8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s. </p>
| + | |
| - | <p>9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. </p>
| + | |
| - | <p>10. To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 µl Buffer EB or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min. </p>
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| - | <h2>Gel Electrophoresis Protocol</h2>
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| - | | + | |
| - | | + | |
| - | <p>1. Measure 0.6g of agarose and add to 60 mL TAE Buffer</p>
| + | |
| - | <p>2. Mix throroughly</p>
| + | |
| - | <p>3. Heat until solution becomes clear</p>
| + | |
| - | <p>4. Allow to cool, then add 30µl of 1mg per ml Ethidium Bromide</p>
| + | |
| - | <p>5. Pour solution into gel tray with comb in place</p>
| + | |
| - | <p>6. Allow to set</p>
| + | |
| - | <p>7. Place in electrophoresis tank and submerge in TAE buffer</p>
| + | |
| - | <p>8. Pipette 10 µl of ladder into left most well</p>
| + | |
| - | <p>9. Perform serial dilution of sample</p>
| + | |
| - | <p>10. Add 9 µl of sample to 1 µl of loading dye and place in wells, noting position of each sample</p>
| + | |
| - | <p>11. Run gel at 90V for 30 minutes</p>
| + | |
| - | <p>12. Visualise using UV light</p>
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| - | </body>
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| - | </html>
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