Team:UT Dallas/Notebook

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<tr><td valign='top' width="250px">
 
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<dl class="accordion">
 
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<dt link='about_utd'>About UTD</dt>
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<h2>Protocols</h2>
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<dd></dd>
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<div id='protocol'>
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          <h3><span></span>Ligation Protocol</h3>
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          <div class="clr"></div>
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          <p></p><li type="disk">Determine insert to vector ratios</li><li type="disk">Calculate the amount of insert needed if 50ng of vector is used (can use different amount of vector)</li><li type="disk">In a PCR tube add the following:<blockquote><li type="circle">50ng of vector</li><li type="circle">Amount of insert based on ratios (calculated in second step)</li><li type="circle">2uL of buffer</li><li type="circle">2uL of DNA ligase</li><li type="circle">Amount of water to bring total volume to 20uL</li></blockquote></li><li type="disk">Incubate overnight at 14 degrees Celsius<p></p></li><p>Note: We used T4 DNA ligase and buffer from NEB</p>
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          <h3><span></span>Gel Purification Protocol (QIAquick Gel Extraction Kit)</h3>
 +
          <div class="clr"></div>
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          <p></p><li type="disk">Excise DNA fragment from the agarose gel with a clean, sharp scalpel</li><li type="disk">Weigh the gel slice in a microcentrifuge tube.</li><li type="disk">Add 3 volumes of Buffer QG to 1 volume of gel (100mg~100uL)</li><li type="disk">Incubate at 50 degrees Celsius for 10 min (until the gel slice has completely dissolved)</li><li type="disk">After the gel slice has dissolved completely, check that the color of the mixture is yellow</li><li type="disk">Apply the sample to a QIAquick column, and centrifuge for 1 min</li><li type="circle"><blockquote>Maximum volume of the column is 800uL. For samples larger than this, simply load and spin again.</blockquote></li><li type="disk">Discard flow-through and place QIAquick column back in the same collection tube</li><li type="disk">To wash, add 750uL of Buffer PE to column and centrifuge for 1 min.</li><li type="disk">Discard the flow-through and centrifuge for additional 1 min. at 13,000rpm</li><li type="disk">Place QIAquick column into a clean 1.5 mL microcentrifuge tube</li><li type="disk">To elute DNA, add 50uL of Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min. and then centrifuge the column for 1 min.<p></p></li>
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          <h3><span></span>Gel Electrophoresis Protocol</h3>
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          <div class="clr"></div>
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          <p></p><li type="disk">Making a 1% agarose gel<blockquote><li type="circle">100mL 1X TBE buffer</li><li type="circle">1g agarose</li><li type="circle">microwave until agarose dissolves</li><li type="circle">let mixture cool</li><li type="circle">when cool add 8-10uL ethidium bromide</li><li type="circle">stir gently, let cool</li><li type="circle">pour into plate with comb already in place</li><li type="circle">let harden</li></blockquote></li><li type="disk">Using the gel<blockquote><li type="circle">Add loading buffer to DNA (for 100uL DNA, add 20uL loading buffer)</li><li type="circle">Load 2uL of DNA ladder into the gel</li><li type="circle">Load DNA into the gel</li><li type="circle">Run at 130V for 30min-1hr<p></p></li></blockquote>
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          <h3><span></span>Digestion Protocol</h3>
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          <div class="clr"></div>
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          <p></p></li><li type="disk">Using a microcentrifuge tube add the following:<blockquote><li type="circle">~3000-5000 ng of DNA</li><li type="circle">10uL Buffer 4</li><li type="circle">10uL BSA</li><li type="circle">5uL of appropriate enzyme (if doing a double digest, use 5 uL of both enzymes)</li><li type="circle">Amount of H2O needed to make final volume 100uL</li></blockquote></li><li type="disk">Incubate at 37 degrees Celsius for 1hr and 30min<p></p></li><p>Note: We used the following enzymes from NEB: EcoRI-HF, PstI-HF, SpeI, and XbaI. All of which can be double digested with each other using Buffer 4.</p>
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          <h3><span></span>Preparing LB+Appropriate Antibiotic Protocol</h3>
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          <div class="clr"></div>
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          <p></p><li type="disk">200 mL LB broth</li><li type="disk">Autoclave<blockquote><li type="circle">Put control thermometer in H2O (from the sink)</li><li type="circle">Select vented container mode (Do Not Change Program)</li></blockquote></li><li type="disk">Let cool to 50 degrees Celsius</li><li type="disk">Add antibiotic (50-100 ug/mL) (10 mg total)<blockquote><li type="circle">Weigh on paper</li><li type="circle">Add to 0.5 mL DI H2O</li><li type="circle">Add to LB mixture when cool enough</li></blockquote></li><li type="disk">Store at 4 degrees Celsius<p></p></li>
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          <h3><span></span>Preparing Agar Plates Protocol (Makes 12 (15mm) Plates)</h3>
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          <div class="clr"></div>
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          <p></p><li type="disk">300 mL DI H2O + 11 g LB agar</li><li type="disk">Autoclave<blockquote><li type="circle">Put control thermometer in H2O (from the sink)</li><li type="circle">Select vented container mode (Do Not Change Program)</li></blockquote></li><li type="disk">Mix well after autoclaving; let cool to 50 degrees Celsius</li><li type="disk">Add antibiotic (50 to 100 µg/mL) (15 mg total)<blockquote><li type="circle">Weigh on paper</li><li type="circle">Add to 0.5 mL DI H2O</li><li type="circle">Add to LB mixture when cool enough</li></blockquote></li><li type="disk">Plate<blockquote><li type="circle">Under flame open lids of all plates</li><li type="circle">Slowly pour agar into plate, avoiding bubbles, when it touches all edges stop pouring</li><li type="circle">Let sit under flames until gel solidifies</li><li type="circle">Replace lids on plates</li></blockquote></li><li type="disk">Store upside down at 4 degrees Celsius<p></p></li>
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          <h3><span></span>Preparing Competent Cells Protocol</h3>
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          <div class="clr"></div>
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          <p></p><li type="disk">Place 1 colony in 5 mL of LB (with antibiotics if appropriate) Grow overnight at 37 degrees Celsius and 200-300 rpm</li><li type="disk">Inoculate 0.25 mL of the overnight strain into 25 mL of LB</li><li type="disk">Shake at 37oC until the OD650 is 0.6-0.7</li><li type="disk">Harvest cells and resuspend in 12.5 mL ice cold 0.1M MgCl2</li><li type="disk">Harvest immediately and resuspend in 7.5 mL cold 0.1M CaCl2</li><li type="disk">Leave on ice for 30 minutes. Harvest and resuspend in 2.5 mL cold 0.1M CaCl2</li><li type="disk">Leave on ice for 30 minutes</li><li type="disk">For long term storage, use 0.1M CaCl2 in 15% glycerol at step 6 and store cells at -800 degrees Celsius<p></p></li><p>
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Note: Harvest cells at 5000 rpm for 10 minutes at 4 degrees Celsius</p>
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          <h3><span></span>Miniprep Protocol (from QIAprep Spin Miniprep Kit)</h3>
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          <div class="clr"></div>
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          <p></p><li type="disk">Harvest cells at 5400g 10 minutes 40 degrees Celsius (possibly program 1)</li><li type="disk">Resuspend pelleted bacterial cells in 250 µL Buffer P1 and transfer to a microcentrifuge tube</li><li type="disk">Add 250 µL Buffer P2 and mix thoroughly by inverting the tube 4-6 times</li><li type="disk">Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times</li><li type="disk">Centrifuge for 10 minutes at 13000 rpm (~17900g) in a table-top microcentrifuge</li><li type="disk">Apply the supernatant (from step 4) to the QIA prep spin column by decanting or pipetting</li><li type="disk">Centrifuge for 30-60 seconds. Discard the flow-through</li><li type="disk">Wash QIA prep spin column by adding 0.75 mL Buffer PE and centrifuging for 30-60 seconds</li><li type="disk">Discard the flow-through, and centrifuge for 1 minute to remove residual wash buffer</li><li type="disk">To elute DNA, place the QIA prep column in a clean 1.5 mL microcentrifuge tube. Add 50 µL Buffer EB or water to the center of each QIA prep spin column, let stand for 1 minute and centrifuge for 1 minute.
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<p></p></li>
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          <h3><span></span>Preparing Glycerol Stock Protocol</h3>
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          <div class="clr"></div>
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          <p></p><li type="disk">Add 150 µL of 50% glycerol to 350 µL of cells</li><li type="disk">Place in -80C freezer<p></p></li>
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          <h3><span></span>Transformation Protocol</h3>
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          <div class="clr"></div>
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          <p></p><li type="disk">With a pipette tip, punch a hole through the foil cover of the DNA plate</li><li type="disk">Add 10 µL of DI water</li><li type="disk">Thaw competent cells on ice</li><li type="disk">Add 1-2 µL of resuspend DNA and 50 µL of thawed competent cells to labeled tubes</li><li type="disk">Incubate the cells on ice for 30 minutes</li><li type="disk">Heat shock the cells at 42 degrees Celsius for 45 sec</li><li type="disk">Incubate the cells on ice for 2 minutes</li><li type="disk">Under flame, add 450 µL SOC broth</li><li type="disk">Incubate at 37 degrees Celsius for 1 hour while rotating or shaking at 300rpm</li><li type="disk">Spread cells on appropriate antibiotic LB plates (usually 100 µL)</li><li type="disk">Incubate at 37 degrees Celsius for 18-24 hours</li><li type="disk">Take a colony, put in 3 mL of LB + appropriate antibiotic</li><li type="disk">Use resulting culture to miniprep DNA and make your own glycerol stock<p></p></li>
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<dt>Students</dt>
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          <h3><span></span>Point mutation Protocol</h3>
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<dd>
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          <div class="clr"></div>
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<ul>
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          1) Create reaction mixture
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<li><a href='#' onclick="show_info('alecspin')">Alec Spin</a></li>
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    <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5 ul 10x buffer
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<li><a href='#' onclick="show_info('student_name');return false">Student 2</a></li>
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          <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;10-100 ng DNA
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<li><a href='#' onclick="show_info('student_name');return false">Student 3</a></li>
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          <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1 ul of foward primer
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<li><a href='#' onclick="show_info('student_name');return false">Student 4</a></li>
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          <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1 ul of reverse primer
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</ul>  
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          <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1 ul of dNTP'S
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</dd>
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          <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1.5 ul of Quik Solution reagent
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          <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Bring to 50 ul with NF-H20
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          <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;*Then add 1ul Quik Change Lightning Enzyme
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  <br>2)Run thermo-cycler (program--mutate)
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          <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1 cycle: @ 95C 2 minutes
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          <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;18 cycles:
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                <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;a) 95C x 20 seconds
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                <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;b) 60C x 10 seconds
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                <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;c) 68C x 30 seconds/kb per plasmid length
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          <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1 cycle: 68C 5 minutes
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  <br>3) Then add 2 ul of DpnI enzyme directly to each amplification reaction
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  <br>4) Pipette up &amp; down several times
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  <br>5) Incubate @ 37C x 5 minutes to digest the parent DNA (cuts methylated dna)
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          <br>6)Then transform.
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</div>
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<div id='calender'>
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<br>
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<h2>Calendar</h2>
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<div class='remote_content' style="padding-top:1px">
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<div id="july"> <a href="#" onclick="getData('week1');return false;" class="week_link">Week 1 </a>
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<br><a href="#" onclick="getData('week2');return false;" class="week_link">Week 2 </a>
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<br><a href="#" onclick="getData('week3');return false;" class="week_link">Week 3 </a>
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<br><a href="#" onclick="getData('week4');return false;" class="week_link">Week 4 </a> </div>
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<div id="august"><a href="#" onclick="getData('week5');return false;" class="week_link">Week 5 </a>
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<br><a href="#" onclick="getData('week6');return false;" class="week_link">Week 6 </a>
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<br><a href="#" onclick="getData('week7');return false;" class="week_link">Week 7 </a>
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<br><a href="#" onclick="getData('week8');return false;" class="week_link">Week 8 </a> </div>
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<div id="sept"> <a href="#" onclick="getData('week9');return false;" class="week_link">Week 9 </a>
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<br><a href="#" onclick="getData('week10');return false;" class="week_link">Week 10 </a>
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<br><a href="#" onclick="getData('week11');return false;" class="week_link">Week 11 </a>
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<br><a href="#" onclick="getData('week12');return false;" class="week_link">Week 12 </a> </div>
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<div style="margin-top:150px;">
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<div id="results" style="margin-bottom:15px">
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<p class="c2"><span>7/5/2013 </span></p><p class="c2"><span>Transformation</span></p><p class="c2"><span>[GFP] Kit plate 5. Well 14k. Backbone pSB1A2. Resistance A. i5-5-1</span></p><p class="c2"><span>[LacI] Kit plate 5. Well 20. Backbone pSB1A2. Resistance A. i5-5-2</span></p><p class="c1"><span></span></p><p class="c2"><span>7/6/2013</span></p><p class="c2"><span>Colony Picking</span></p><p class="c2"><span>I5-5-1 ? &nbsp;2 colonies ? picked 2 ? i5-5-3</span></p><p class="c2"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; ? i5-5-4</span></p><p class="c2"><span>I5-5-2 ? 0 colonies</span></p><p class="c1"><span></span></p><p class="c2"><span>7/7/2013</span></p><p class="c2"><span>Mini Prep</span></p><p class="c2"><span>I5-5-3: 67.4 ng/ul&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;[GFP]</span></p><p class="c2"><span>I5-5-4: 98.7 ng/ul&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;[GFP]</span></p><p class="c2"><span>I5-5-5: 408.7 ng/ul&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;[+ confiol]</span></p><p class="c1"><span></span></p><p class="c2"><span>Test Digestion</span></p><a href="#" name="d2fd616f7186be08d9c7705395c2136d5ae85998"></a><a href="#" name="0"></a><table class="c12" cellpadding="0" cellspacing="0"><tbody><tr class="c37"><td class="c68"><p class="c1 c4"><span></span></p></td><td class="c68"><p class="c4 c2"><span>I5-5-3</span><span class="c97">1</span></p></td><td class="c68"><p class="c4 c2"><span>I5-5-4</span><span class="c97">2</span></p></td></tr><tr class="c56"><td class="c68"><p class="c4 c2"><span>DNA</span></p></td><td class="c68"><p class="c4 c2"><span>4</span></p></td><td class="c68"><p class="c4 c2"><span>2</span></p></td></tr><tr class="c37"><td class="c68"><p class="c4 c2"><span>BSA</span></p></td><td class="c68"><p class="c4 c2"><span>1</span></p></td><td class="c68"><p class="c4 c2"><span>1</span></p></td></tr><tr class="c56"><td class="c68"><p class="c4 c2"><span>Buffer 4</span></p></td><td class="c68"><p class="c4 c2"><span>1</span></p></td><td class="c68"><p class="c4 c2"><span>1</span></p></td></tr><tr class="c37"><td class="c68"><p class="c4 c2"><span>EcoRI</span></p></td><td class="c68"><p class="c4 c2"><span>1</span></p></td><td class="c68"><p class="c4 c2"><span>1</span></p></td></tr><tr class="c56"><td class="c68"><p class="c4 c2"><span>Pst</span></p></td><td class="c68"><p class="c4 c2"><span>1</span></p></td><td class="c68"><p class="c4 c2"><span>1</span></p></td></tr><tr class="c56"><td class="c68"><p class="c4 c2"><span>H</span><span class="c107">2</span><span>O</span></p></td><td class="c68"><p class="c4 c2"><span>2</span></p></td><td class="c68"><p class="c4 c2"><span>4</span></p></td></tr><tr class="c56"><td class="c68"><p class="c1 c4"><span></span></p></td><td class="c68"><p class="c4 c2"><span>10</span></p></td><td class="c68"><p class="c4 c2"><span>10</span></p></td></tr></tbody></table>
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<dt>Advisors</dt>
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</div>
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<dd>
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<ul>
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<li><a href='#' onclick="show_info('Advisor_name');return false">Advisor 1</a></li>
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<li><a href='#' onclick="show_info('Advisor_name');return false">Advisor 2</a></li>
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<li><a href='#' onclick="show_info('Advisor_name');return false">Advisor 3</a></li>
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<li><a href='#' onclick="show_info('Advisor_name');return false">Advisor 4</a></li>
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</ul>
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</dd>
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</dl>
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<span class="title_spans">About UT Dallas</span><br><br>
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<img src="https://static.igem.org/mediawiki/2013/8/81/UTDallas_Logo.png" style="float:right;margin-left:10px;margin-bottom:5px">
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The University of Texas at Dallas (also referred to as UT Dallas or UTD) is a public research university in the University of Texas System. The main campus is in Richardson, Texas, Telecom Corridor, 18 miles (29 km) north of downtown Dallas. The institution, established in 1961 as the Graduate Research Center of the Southwest and later renamed the Southwest Center for Advanced Studies (SCAS), began as a research arm of Texas Instruments. In 1969 the founders bequeathed SCAS to the state of Texas and Governor Preston Smith signed the bill officially creating the University of Texas at Dallas.
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<br><br>
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UTD offers over 133 academic programs across its seven schools and hosts more than 50 research centers and institutes. With a number of interdisciplinary degree programs, its curriculum is designed to allow study that crosses traditional disciplinary lines and to enable students to participate in collaborative research labs. Entering freshmen average math and critical reading SAT scores are among the highest of the public universities in Texas and 1270 for 2012. The Carnegie Foundation classifies UT Dallas as a "comprehensive doctoral research university" and a "high research activity institution". Research projects include the areas of space science, bioengineering, cybersecurity, nanotechnology, and behavioral and brain sciences.
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<br><br>
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UT Dallas owns approximately 710 acres (2.9 km2) of generally contiguous land in Richardson, Texas consisting of 445 acres (1.80 km2) for campus development and another 265 acres (1.07 km2) adjacent to the main campus. From 2007 the university has added or started construction on more than 1,000,000 square feet (93,000 m2) of new facilities including the first academic structure in Texas to be rated a LEED Platinum facility. A $30-million campus landscape enhancement was completed in late 2010 and an additional $15-million campus landscape development phase is scheduled to begin in 2013.
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<br><br>
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The school has a Division III athletics program in the American Southwest Conference and fields 13 intercollegiate teams. Student activities include more than 225 registered organizations and students provided more than 20,000 volunteer hours at community agencies in 2010. The university has a nationally recognized debate team, recruits worldwide for its chess team and the only school in Texas to field teams in all three of the major pre-law competitions. For the spring 2013 commencement the university granted 1,557 bachelor's degrees, 1,380 master's degrees and 87 PhDs for a total of 3,024 degrees.
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Latest revision as of 00:27, 16 October 2013

Protocols

Ligation Protocol

  • Determine insert to vector ratios
  • Calculate the amount of insert needed if 50ng of vector is used (can use different amount of vector)
  • In a PCR tube add the following:
  • 50ng of vector
  • Amount of insert based on ratios (calculated in second step)
  • 2uL of buffer
  • 2uL of DNA ligase
  • Amount of water to bring total volume to 20uL
  • Incubate overnight at 14 degrees Celsius

    • Note: We used T4 DNA ligase and buffer from NEB

    Gel Purification Protocol (QIAquick Gel Extraction Kit)

  • Excise DNA fragment from the agarose gel with a clean, sharp scalpel
  • Weigh the gel slice in a microcentrifuge tube.
  • Add 3 volumes of Buffer QG to 1 volume of gel (100mg~100uL)
  • Incubate at 50 degrees Celsius for 10 min (until the gel slice has completely dissolved)
  • After the gel slice has dissolved completely, check that the color of the mixture is yellow
  • Apply the sample to a QIAquick column, and centrifuge for 1 min
  • Maximum volume of the column is 800uL. For samples larger than this, simply load and spin again.
  • Discard flow-through and place QIAquick column back in the same collection tube
  • To wash, add 750uL of Buffer PE to column and centrifuge for 1 min.
  • Discard the flow-through and centrifuge for additional 1 min. at 13,000rpm
  • Place QIAquick column into a clean 1.5 mL microcentrifuge tube
  • To elute DNA, add 50uL of Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min. and then centrifuge the column for 1 min.

    • Gel Electrophoresis Protocol

  • Making a 1% agarose gel
  • 100mL 1X TBE buffer
  • 1g agarose
  • microwave until agarose dissolves
  • let mixture cool
  • when cool add 8-10uL ethidium bromide
  • stir gently, let cool
  • pour into plate with comb already in place
  • let harden
  • Using the gel
  • Add loading buffer to DNA (for 100uL DNA, add 20uL loading buffer)
  • Load 2uL of DNA ladder into the gel
  • Load DNA into the gel
  • Run at 130V for 30min-1hr

    • Digestion Protocol

  • Using a microcentrifuge tube add the following:
  • ~3000-5000 ng of DNA
  • 10uL Buffer 4
  • 10uL BSA
  • 5uL of appropriate enzyme (if doing a double digest, use 5 uL of both enzymes)
  • Amount of H2O needed to make final volume 100uL
  • Incubate at 37 degrees Celsius for 1hr and 30min

    • Note: We used the following enzymes from NEB: EcoRI-HF, PstI-HF, SpeI, and XbaI. All of which can be double digested with each other using Buffer 4.

    Preparing LB+Appropriate Antibiotic Protocol

  • 200 mL LB broth
  • Autoclave
  • Put control thermometer in H2O (from the sink)
  • Select vented container mode (Do Not Change Program)
  • Let cool to 50 degrees Celsius
  • Add antibiotic (50-100 ug/mL) (10 mg total)
  • Weigh on paper
  • Add to 0.5 mL DI H2O
  • Add to LB mixture when cool enough
  • Store at 4 degrees Celsius

    • Preparing Agar Plates Protocol (Makes 12 (15mm) Plates)

  • 300 mL DI H2O + 11 g LB agar
  • Autoclave
  • Put control thermometer in H2O (from the sink)
  • Select vented container mode (Do Not Change Program)
  • Mix well after autoclaving; let cool to 50 degrees Celsius
  • Add antibiotic (50 to 100 µg/mL) (15 mg total)
  • Weigh on paper
  • Add to 0.5 mL DI H2O
  • Add to LB mixture when cool enough
  • Plate
  • Under flame open lids of all plates
  • Slowly pour agar into plate, avoiding bubbles, when it touches all edges stop pouring
  • Let sit under flames until gel solidifies
  • Replace lids on plates
  • Store upside down at 4 degrees Celsius

    • Preparing Competent Cells Protocol

  • Place 1 colony in 5 mL of LB (with antibiotics if appropriate) Grow overnight at 37 degrees Celsius and 200-300 rpm
  • Inoculate 0.25 mL of the overnight strain into 25 mL of LB
  • Shake at 37oC until the OD650 is 0.6-0.7
  • Harvest cells and resuspend in 12.5 mL ice cold 0.1M MgCl2
  • Harvest immediately and resuspend in 7.5 mL cold 0.1M CaCl2
  • Leave on ice for 30 minutes. Harvest and resuspend in 2.5 mL cold 0.1M CaCl2
  • Leave on ice for 30 minutes
  • For long term storage, use 0.1M CaCl2 in 15% glycerol at step 6 and store cells at -800 degrees Celsius

    • Note: Harvest cells at 5000 rpm for 10 minutes at 4 degrees Celsius

    Miniprep Protocol (from QIAprep Spin Miniprep Kit)

  • Harvest cells at 5400g 10 minutes 40 degrees Celsius (possibly program 1)
  • Resuspend pelleted bacterial cells in 250 µL Buffer P1 and transfer to a microcentrifuge tube
  • Add 250 µL Buffer P2 and mix thoroughly by inverting the tube 4-6 times
  • Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times
  • Centrifuge for 10 minutes at 13000 rpm (~17900g) in a table-top microcentrifuge
  • Apply the supernatant (from step 4) to the QIA prep spin column by decanting or pipetting
  • Centrifuge for 30-60 seconds. Discard the flow-through
  • Wash QIA prep spin column by adding 0.75 mL Buffer PE and centrifuging for 30-60 seconds
  • Discard the flow-through, and centrifuge for 1 minute to remove residual wash buffer
  • To elute DNA, place the QIA prep column in a clean 1.5 mL microcentrifuge tube. Add 50 µL Buffer EB or water to the center of each QIA prep spin column, let stand for 1 minute and centrifuge for 1 minute.

    • Preparing Glycerol Stock Protocol

  • Add 150 µL of 50% glycerol to 350 µL of cells
  • Place in -80C freezer

    • Transformation Protocol

  • With a pipette tip, punch a hole through the foil cover of the DNA plate
  • Add 10 µL of DI water
  • Thaw competent cells on ice
  • Add 1-2 µL of resuspend DNA and 50 µL of thawed competent cells to labeled tubes
  • Incubate the cells on ice for 30 minutes
  • Heat shock the cells at 42 degrees Celsius for 45 sec
  • Incubate the cells on ice for 2 minutes
  • Under flame, add 450 µL SOC broth
  • Incubate at 37 degrees Celsius for 1 hour while rotating or shaking at 300rpm
  • Spread cells on appropriate antibiotic LB plates (usually 100 µL)
  • Incubate at 37 degrees Celsius for 18-24 hours
  • Take a colony, put in 3 mL of LB + appropriate antibiotic
  • Use resulting culture to miniprep DNA and make your own glycerol stock

    • Point mutation Protocol

    1) Create reaction mixture
            5 ul 10x buffer
            10-100 ng DNA
            1 ul of foward primer
            1 ul of reverse primer
            1 ul of dNTP'S
            1.5 ul of Quik Solution reagent
            Bring to 50 ul with NF-H20
            *Then add 1ul Quik Change Lightning Enzyme
    2)Run thermo-cycler (program--mutate)
            1 cycle: @ 95C 2 minutes
            18 cycles:
                    a) 95C x 20 seconds
                    b) 60C x 10 seconds
                    c) 68C x 30 seconds/kb per plasmid length
            1 cycle: 68C 5 minutes
    3) Then add 2 ul of DpnI enzyme directly to each amplification reaction
    4) Pipette up & down several times
    5) Incubate @ 37C x 5 minutes to digest the parent DNA (cuts methylated dna)
    6)Then transform.

    Calendar

    7/5/2013

    Transformation

    [GFP] Kit plate 5. Well 14k. Backbone pSB1A2. Resistance A. i5-5-1

    [LacI] Kit plate 5. Well 20. Backbone pSB1A2. Resistance A. i5-5-2

    7/6/2013

    Colony Picking

    I5-5-1 ?  2 colonies ? picked 2 ? i5-5-3

                                            ? i5-5-4

    I5-5-2 ? 0 colonies

    7/7/2013

    Mini Prep

    I5-5-3: 67.4 ng/ul        [GFP]

    I5-5-4: 98.7 ng/ul        [GFP]

    I5-5-5: 408.7 ng/ul        [+ confiol]

    Test Digestion

    I5-5-31

    I5-5-42

    DNA

    4

    2

    BSA

    1

    1

    Buffer 4

    1

    1

    EcoRI

    1

    1

    Pst

    1

    1

    H2O

    2

    4

    10

    10

    Retrieved from "http://2013.igem.org/Team:UT_Dallas/Notebook"