Template:Team:Sheffield/Content:LabProtocols

From 2014.igem.org

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<h1 class="subPageTitle">Lab Protocols</h1>
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<h1 class="subPageTitle">Dry Lab Journal</h1>
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At the start of our project, we undertook a week of boot camp – yes, it was as intense as it sounds! With all team members coming from a range of backgrounds with different abilities, we thought that this would be a good idea to allow everyone to be on the same page with the same basic level of understanding in each component of the project.
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<!-- section templates
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<div class="pageSection1">
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<div class="pageSection1">
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<div class="sectionHeading">
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<div class="sectionHeading">
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<h2>Protocol 1: Produce LB Broth</h2>
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<h2></h2>
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</div>
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<div class="sectionContent">
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<h3>Materials and Equipment</h3>
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-
LB broth mix, sterile water, weighing scales, weighing boat, conical flask, measuring cylinder.
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<h3>Time</h3>
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Prep: 5 minutes<br/>
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Run: 2 hours
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<h3>Procedure</h3>
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<ol>
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<li>Weigh out 2g of LB broth mix in a weighing boat and add into a 250ml conical flask.</li>
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<li>Measure 100ml sterile water into a measuring cylinder and dispense into the same conical flask.</li>
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<li>Swirl.</li>
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<li>Stopper the flask with cotton wool and foil.</li>
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<li>Label on autoclave tape. Format 'iGEM, Room Number'.</li>
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<li>Move the flask(s) to the autoclave to be sterilised.</li>
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</ol>
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</div>
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</div>
</div>
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<div class="sectionContent">
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<div class="pageSection2">
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<h3></h3>
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<div class="sectionHeading">
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<h2>Protocol 2: Produce LB Agar</h2>
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</div>
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<div class="sectionContent">
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<h3>Materials and Equipment</h3>
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-
LB Agar mix, sterile water, weighing scales, weighing boat, conical flask, measuring cylinder.
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<h3>Time</h3>
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-
Prep: 5 minutes<br/>
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-
Run: 2 hours
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<h3>Procedure</h3>
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-
<ol>
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-
<li>Weigh out 3.5g of LB agar mix in a weighing boat and add into a conical flask.</li>
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<li>Measure 100ml sterile water into a measuring cylinder and dispense into the same conical flask.</li>
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<li>Swirl.</li>
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-
<li>Stopper the flask with cotton wool and foil.</li>
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-
<li>Label on autoclave tape. Format 'iGEM, Room Number'.</li>
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-
<li>Move the flask(s) to the autoclave to be sterilised.</li>
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-
</ol>
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</div>
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</div>
</div>
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</div>
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<div class="pageSection1">
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<div class="sectionHeading">
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<div class="pageSection2">
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<h2>Protocol 3: Make overnight starter cultures</h2>
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<div class="sectionHeading">
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</div>
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<h2></h2>
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<div class="sectionContent">
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<h3>Materials and Equipment</h3>
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-
Stripette, bunsen burner, sterile loop, falcon tubes, LB broth.
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<h3>Time</h3>
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Prep: 10 minutes<br/>
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Run: 16 hours
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<h3>Procedure</h3>
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<ol>
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<li>Use a stripette to take 5ml of LB broth from a 250ml conical flask.</li>
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<li>Dispense into a falcon tube.</li>
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<li>Sterilise a metal loop in a flame.</li>
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<li>Take a culture from an agar plate using the sterile loop and put into one of the falcon tubes.</li>
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<li>Agitate.</li>
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<li>Replicate this using scrapings from a clean agar plate and a fresh tube to use as a positive control.</li>
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<li>Put the tubes into the incubator overnight (37c, 150rpm) to grow up the cultures.</li>
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</ol>
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</div>
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</div>
</div>
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<div class="sectionContent">
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<div class="pageSection2">
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<h3></h3>
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<div class="sectionHeading">
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<h2>Protocol 4: Generate chemically competent E. Coli</h2>
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</div>
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<div class="sectionContent">
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<h3>Materials and Equipment</h3>
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LB broth, starter culture, P1000, P200, pipette tips, incubator, cuvettes, spectrophotometer, Virkon, falcon tubes, ice, weighing scales, centrifuge, CaCl2, 20% glycerol, stripette, eppendorf tubes and -80°C freezer.
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<h3>Time</h3>
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Prep: 40 minutes<br/>
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Run: 5 hours
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<h3>Procedure</h3>
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<ol>
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<li>Grow cells
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<ol>
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<li>Take 1ml starter culture and add to 100ml LB broth.</li>
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<li>Incubate at 37°C, 150rpm.</li>
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<li>Check every hour by testing the optical density at 600nm (OD) using a spectrophotometer to determine whether enough cells are present in the culture.</li>
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<li>0.600 OD is ideal, this is the point at which the cells are in the exponential growth phase.</li>
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<li>Take 1ml of culture into a cuvette to measure; dispose of this after use in Virkon.</li>
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</ol>
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</li>
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<li>Remove cells
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<ol>
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<li>Pour 30ml aliquots from the flasks into falcon tubes; 3 tubes per flask.</li>
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<li>Put tubes on ice for approx 10mins; all equipment used from this point on must be cold e.g. pipette tips.</li>
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<li>Weigh the falcon tubes and pair together similar weight tubes for balance in the centrifuge; tubes paired together must weigh within 0.5g of each other.</li>
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<li>Spin at 4°C, 4000rpm for 5mins.</li>
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<li>After the tubes have all been spun, pour off the supernatant to remove the LB broth, leaving cells in a pellet. Put tubes back on the ice.</li>
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</ol>
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</li>
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<li>Make cells competent
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<ol>
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<li>Add 1ml of CaCl2 to the cells, use the pipette to pull the liquid and cells up and down to resuspend.</li>
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<li>Once resuspended, add another 14ml of CaCl2; 15ml total volume.</li>
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<li>Put back on ice for approx. 10mins to allow cells to acclimatise at the temperature with the CaCl2.</li>
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<li>Re-weigh and pairs tubes again for balance.</li>
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<li>Spin again at 4°C, 4000rpm for 5mins.</li>
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<li>After tubes have been spun, leave on ice for 5mins. Pour off the supernatant?</li>
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</ol>
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</li>
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<li>Aliquot
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<ol>
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<li>Add 600μl of 20% glycerol to each falcon tube.</li>
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<li>Label eppendorf tubes.</li>
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<li>Aliquot 200μl from each falcon tube into eppendorf tubes (3 per falcon).</li>
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<li>Freeze at -80°C.</li>
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</ol>
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</li>
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</ol>
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</div>
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</div>
</div>
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</div>
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<div id="protocol5" class="pageSection1">
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-->
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<div class="sectionHeading">
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<h2>Protocol 5: Mini-Prep</h2>
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<div class="pageSection2">
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</div>
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<div class="sectionHeading">
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<div class="sectionContent">
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<h2>Training Week</h2>
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<h3>Materials and Equipment</h3>
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-
Ice, starter cultures, mini-prep kit, centrifuge, P100 pipette, weighing scale, Virkon.
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-
<h3>Time</h3>
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-
Prep: ?<br/>
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Run: ?
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<h3>Procedure</h3>
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-
<ol>
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<li>Extract cells
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-
<ol>
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<li>Match up weights of falcon tubes so they are paired within 0.5g.</li>
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<li>Spin down starter cultures in a centrifuge; 5mins, 4°C, 4000rpm.</li>
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<li>Pour off supernatant into Virkon.</li>
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</ol>
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</li>
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<li>Resuspend cells
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<ol>
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<li>Add 250μl of P1 resuspension buffer to each falcon tube using P1000.</li>
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<li>Resuspend the cells.</li>
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<li>Use pipette to move suspension into separate, labelled eppendorf tubes.</li>
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</ol>
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</li>
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<li>Lyse cells
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<ol>
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<li>Add 250μl of P2 buffer to each eppendorf tube.</li>
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<li>This will lyse cells - a blue colour will indicate they have been lysed.</li>
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<li>Do not leave for more than 5mins.</li>
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-
</ol>
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</li>
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<li>Neutralise cells
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-
<ol>
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<li>Add 350μl of N3 neutralisation buffer.</li>
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<li>Once the reaction is complete, the liquid will turn clear/white.</li>
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-
</ol>
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</li>
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<li>Purify DNA
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-
<ol>
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-
<li>Spin down the cells in a centrifuge; 10 mins, 17000g, 4°C.</li>
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-
<li>Pour supernatant into mini-prep columns.</li>
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<li>Centrifuge columns for 1 min, 17000g, 4°C.</li>
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<li>Discard flow through.</li>
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<li>Add 500μl of PB buffer to each column.</li>
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<li>Centrifuge columns for 1min, 17000g, 4°C.</li>
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<li>Discard flow through.</li>
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<li>Add 750μl PE buffer to each column.</li>
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<li>Centrifuge columns for 1min, 17000g, 4°C.</li>
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<li>Pour off supernatent.</li>
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<li>Centrifuge columns for 1min, 17000g, 4°C.</li>
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<li>Discard bottom of the mini-prep.</li>
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-
<li>Move column to a new labelled eppendorf.</li>
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-
<li>Add 50μl of elution buffer.</li>
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<li>Centrifuge columns for 1min, 17000g, 4°C.</li>
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<li>Discard column.</li>
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-
<li>Immediately place on ice; store in the B56 sewer sample freeze.</li>
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-
</ol>
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</li>
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</ol>
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-
</div>
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</div>
</div>
 +
<div class="sectionContent">
 +
<h3>Monday 16th June</h3>
 +
<ul>
 +
<li>Laboratory Orientation and Safety Training</li>
 +
<li>Laboratory Techniques 1</li>
 +
<ul>
 +
<li>Pipetting, LB Broth, LB agar and Overnight Cultures</li>
 +
</ul>
 +
<li>Project Expectations Discussion</li>
 +
<ul>
 +
<li>Medal criteria, weekly meetings to discuss objectives and results, personal logs of what and how things have been done</li>
 +
</ul>
 +
</ul>
 +
 +
<h3>Tuesday 17th June</h3>
 +
<ul>
 +
<li>Introduction of Policy and Practices</li>
 +
<li>Discussion of Social Implications of Synthetic Biology</li>
 +
<li>Genome 101</li>
 +
<li>Laboratory Techniques 2</li>
 +
<ul>
 +
<li>Plasmid DNA extraction, PCR and Chemical Transformation</li>
 +
</ul>
 +
</ul>
 +
<h3>Wednesday 18th June</h3>
 +
<ul>
 +
<li>Introduction to Modelling</li>
 +
<li>BioBricks and the Registry</li>
 +
<li>Methodology in Social Science and Data Collection</li>
 +
</ul>
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<div class="pageSection2">
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<h3>Thursday 19th June</h3>
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<div class="sectionHeading">
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<ul>
-
<h2>Protocol 6: Run a gel</h2>
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<li>Oxford Meet Up</li>
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</div>
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<li>Wiki and Team Identity Discussion</li>
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<div class="sectionContent">
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</ul>
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<h3>Materials and Equipment</h3>
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-
Agarose mix, TAE buffer, Flask, Microwave, Ethidium bromide, P10, Tips, Autoclave tape, Gel tray, Comb, Buffer tray, Loading buffer, Dna, Eppendorf, Transilluminator.
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<h3>Friday 20th June</h3>
-
<h3>Time</h3>
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<ul>
-
Prep: 30 mintues<br/>
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<li>Project Presentation to the Chemical and Biological Engineering Department at the University of Sheffield</li>
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Run: 1 hour
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<li>Weekly Report and Feedback</li>
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<h3>Procedure</h3>
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</ul>
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<ol>
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-
<li>Make up a 1% agarose gel
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-
<ol>
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-
<li>Weigh out 0.4g agarose.</li>
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-
<li>Measure 40ml TAE buffer.</li>
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-
<li>Add both into a 250ml conical flask.</li>
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-
<li>Swirl.</li>
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<li>Microwave on full for 2 minute, swirling at intervals to ensure all the agarose has dissolved.</li>
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<li>Run side of flask under tap to cool, until comfortable to hold in a gloved hand.</li>
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-
</ol>
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</li>
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-
<li>Prepare the gel
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-
<ol>
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-
<li>Add 1μl ethidium bromide to the conical flask containing the melted agarose.</li>
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<li>Pour this solution smoothly into a gel tray (sealed with autoclave tape) and add a comb which allows for either 8 or 13 DNA samples to be run at once.</li>
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<li>Leave the gel to set for approximately 15 - 30 minutes.</li>
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<li>Once the gel is set remove the comb and autoclave tape.</li>
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<li>Place the gel tray inside the buffer tray and fill the remaining space with TAE buffer.</li>
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</ol>
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</li>
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<li>Load 'checking' samples
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<ol>
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<li>Load 5μl of 1kn ladder into the first well of the gel.</li>
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<li>Pipette 2μl 5x loading buffer onto a sheet of parafilm.</li>
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<li>pipette 8μl DNA onto the loading buffer and pipette up and down to mix.</li>
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<li>Resting the pipette tip on the back of the well, load 8μl of sample.</li>
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<li>Repeat for each sample.</li>
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<li>Run the Gel at 100v for 60 minutes.</li>
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-
</ol>
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</li>
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<li>Load 'extraction' samples
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-
<ol>
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<li>Load 5μl of 1kn ladder into the first well of the gel.</li>
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<li>Add 5x loading buffer into each sample, to give a final ratio of 1:4.</li>
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<li>Resting the pipette tip on the back of the well, load as much sample as possible.</li>
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<li>Repeat for each sample.</li>
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</ol>
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</li>
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<li>Visualise
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-
<ol>
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<li>Remove the agarose gel from the gel tray and place in the trans-illuminator.</li>
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<li>Open UVP (on the computer desktop).</li>
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<li>Select the type of gel and the exposure time.</li>
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<li>Press capture to take an image.</li>
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<li>Save image to the iGEM2014 folder.</li>
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</ol>
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</li>
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</ol>
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</div>
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</div>
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<div class="pageSection1">
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<br>This week was very useful as it allowed us to become familiar with all aspects of the project. As well as this, it was a great icebreaker that helped our team of students, advisors and supervisors to get to know each other better!
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<div class="sectionHeading">
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<h2>Protocol 7: Pouring Plates</h2>
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</div>
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<div class="sectionContent">
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<h3>Materials and Equipment</h3>
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Sterile hood, Flask, Agar mix, dH2O, antibiotic, empty plates, P200, tips.
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<h3>Time</h3>
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Prep: 15 minutes<br/>
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Run: 20 minutes
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<h3>Procedure</h3>
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<ol>
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<li>Use the sterile hood.</li>
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<li>Make up 100ml LB Agar (Protocol 2).</li>
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-
<li>Heat in the microwave as follows:
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-
<ul>
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-
<li>Power setting 4 for 3 minutes.</li>
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-
<li>Wait for 6 minutes.</li>
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<li>Power setting 4 for 2 minutes.</li>
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</ul>
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</li>
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<li>Wait to cool (until the flask is warm but comfortable to hold).</li>
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<li>Add 100μl 1/1000 stock of appropriate antibiotic to the agar.</li>
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<li>Pour 4 plates and leave inside the hood to cool and dry.</li>
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-
</ol>
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</div>
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</div>
</div>
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</div>
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<div class="pageSection2">
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<div class="sectionHeading">
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<div class="pageSection1">
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<h2>Protocol 8: Measure Concentration of DNA with NanoDrop 2000</h2>
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<div class="sectionHeading">
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</div>
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<h2>Week 1</h2>
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<div class="sectionContent">
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-
<h3>Materials and Equipment</h3>
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-
Nanodrop, P10, Tips, dH2O, Paper towel.
+
-
<h3>Time</h3>
+
-
Prep: 5 minutes<br/>
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-
Run: 5 minutes
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-
<h3>Procedure</h3>
+
-
<ol>
+
-
<li>Set up the NanoDrop2000, select “Nucleic Acid”, then “Routine Verification”.</li>
+
-
<li>Spray paper towel with distilled water, then clean metal nodules on metal part and lid.</li>
+
-
<li>Add 1μl of buffer solution to nodule.</li>
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<li>Close lid.</li>
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-
<li>Select 'Blank' on the programme.</li>
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-
<li>Reopen lid.</li>
+
-
<li>Add 2μl of DNA Sample.</li>
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-
<li>Close lid.</li>
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-
<li>Select 'measure' on the programme.</li>
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<li>Take note of the concentration (in ng/ml).</li>
+
-
<li>Clean nodules again with distilled water.</li>
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<li>Repeat for different DNA sample.</li>
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-
</ol>
+
-
</div>
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</div>
</div>
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+
<div class="sectionContent">
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<div class="pageSection1">
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<h3>Monday 23rd June</h3>
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<div class="sectionHeading">
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<ul>
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<h2>Protocol 9: Transforming Cells To Ensure Competency</h2>
+
<li>Collated contact details of potential sponsors</li>
-
</div>
+
<li>Drafter sponsorship request letters for money, equipment and expertise</li>
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<div class="sectionContent">
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</ul>
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<h3>Materials and Equipment</h3>
+
-
Eppendorfs, P1000, P200, P10, Tips, Competent cells, Ligation mix, Ice, Heat block, LB broth, Incubator, Agar plates, Flame, Spreader, Ethanol
+
<h3>Tuesday 24th June</h3>
-
<h3>Time</h3>
+
<ul>
-
Prep: 5 minutes<br/>
+
<li>Continued working towards producing a sponsorship package</li>
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Run: 1 hour
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<li>Reading published papers relating to synthetic biology and iGEM regarding awareness and impressions</li>
-
<h3>Procedure</h3>
+
<li>Set up two meetings: marketing and industry expertise</li>
-
<ol>
+
</ul>
-
<li>Using concentration measure by the nanodrop, calculate the volume of DNA sample required: C1V1 = C2V2.</li>
+
-
<li>Where C2 required is 10ng and V2 is 100μl.</li>
+
<h3>Wednesday 25th June</h3>
-
<li>Use the calculated volume of DNA to transform (Protocol 11) chemically competent cells before plating out.</li>
+
<ul>
-
<li>Calculate the transformation efficiency in colonies per ng of DNA.</li>
+
<li>Mathematica – 4 of our student team members went on a course to learn Mathematica for the modelling aspect of the project</li>
-
</ol>
+
<li>Further reading of policy and practices papers</li>
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</div>
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<li>Worked on team identity in preparation for the marketing meeting</li>
 +
<li>Sponsorship package completed – this can be found here</li>
 +
<li>Translated written protocols into the lab notation</li>
 +
</ul>
 +
 +
<h3>Thursday 26th June</h3>
 +
<ul>
 +
<li>Started planning our Meet Up and contacted teams for potential collaboration</li>
 +
<li>Continued work on the lab notation</li>
 +
<li>Discussed potential research questions to carry out a study within the University</li>
 +
<li>Meeting with industry expert within the University of Sheffield; this provided us with new contacts and a better</li> understanding of the UK sewage system and how it functions. It was also very useful as we began to consider the applications of our project and what we may need to consider.</li>
 +
Mathematica
 +
</ul>
 +
 +
<h3>Friday 27th June</h3>
 +
<ul>
 +
<li>Used XML and PYTHON to try to produce a computer language for the lab notation</li>
 +
<li>Mathematica</li>
 +
<li>Weekly meeting and report</li>
 +
</ul>
</div>
</div>
-
 
+
</div>
-
<div class="pageSection2">
+
-
<div class="sectionHeading">
+
<div class="pageSection2">
-
<h2>Protocol 10: Blunt End Ligation</h2>
+
<div class="sectionHeading">
-
</div>
+
<h2>Week 2</h2>
-
<div class="sectionContent">
+
-
<h3>Materials and Equipment</h3>
+
-
Eppendorfs, P10, tips, Reaction buffer, dH2O, Restricted DNA, T4 Ligase.
+
-
<h3>Time</h3>
+
-
Prep: 10 minutes<br/>
+
-
Run: 10 minutes
+
-
<h3>Procedure</h3>
+
-
<ol>
+
-
<li>Make up eppendorfs containing ligation mix, as shown:
+
-
<ul>
+
-
<li>10μl reaction buffer.</li>
+
-
<li>8μl water.</li>
+
-
<li>2μl Cut DNA.</li>
+
-
<li>1μl Cut plasmid.</li>
+
-
<li>1μl T4 ligase.</li>
+
-
</ul>
+
-
</li>
+
-
<li>Flick to mix and leave for ten minutes.</li>
+
-
</ol>
+
-
</div>
+
</div>
</div>
-
 
+
<div class="sectionContent">
-
<div class="pageSection1">
+
<h3>Monday 30th June</h3>
-
<div class="sectionHeading">
+
<ul>
-
<h2>Protocol 11: Chemical Tranformation</h2>
+
<li>Weekly meeting to set objectives</li>
-
</div>
+
<li>Worked on the interview questions to be used for the University of Sheffield case study</li>
-
<div class="sectionContent">
+
<li>Further research on potential sponsors</li>
-
<h3>Materials and Equipment</h3>
+
<li>Mathematica</li>
-
Eppendorfs, P1000, P200, P10, Tips, Competent cells, Ligation mix, Ice, Heat block, LB broth, Incubator, Agar plates, Flame, Spreader, Ethanol.
+
</ul>
-
<h3>Time</h3>
+
-
Prep: 5 minutes<br/>
+
<h3>Tuesday 1st July</h3>
-
Run: 1 hour 45 minutes
+
<ul>
-
<h3>Procedure</h3>
+
<li>Meeting with advisors to discuss suitability of research question and interviews</li>
-
<ol>
+
<li>Began to consider the ethics needed to go alongside this</li>
-
<li>Take an appropriate number of eppendorfs, each containing 100μl of competent E. coli cells.</li>
+
<li>Mathematica</li>
-
<li>Into each one, pipette 5μl of each ligation mix.</li>
+
</ul>
-
<li>Leave a last one to act as a negative control.</li>
+
-
<li>Put the eppendorfs on ice for 30 minutes.</li>
+
<h3>Wednesday 2nd July</h3>
-
<li>Heat shock eppendorfs at 42°C for 30 seconds.</li>
+
<ul>
-
<li>Put on ice for 2 minutes.</li>
+
<li>Finalised research questions and sent off ethics approval</li>
-
<li>Add 1ml LB broth to each of the eppendorfs and then incubate at 37°C for 60 minutes.</li>
+
<li>Began learning HTML and other mark up languages for the Wiki</li>
-
<li>Remove cells from incubator and spin down at 13000RPM for 60 seconds.</li>
+
</ul>
-
<li>Pour off the supernatant and resuspend the cells.</li>
+
-
<li>Near a flame, pipette each of the remaining cells onto individual plates (prepared earlier).</li>
+
<h3>Thursday 3rd July</h3>
-
<li>Sterilise a glass spreader using ethanol (flamed) and spread cells evenly until the surface of the agar appears dry.</li>
+
<ul>
-
<li>Incubate at 37°C overnight.</li>
+
<li>Team identity discussion on potential project names and logo</li>
-
</ol>
+
<li>Researched assays</li>
-
</div>
+
<li>Wrote COSHH forms</li>
 +
<li>Sent out sponsorship packages</li>
 +
<li>ERASynBio research and application draft</li>
 +
</ul>
 +
 +
<h3>Friday 4th July</h3>
 +
<ul>
 +
<li>Sent out invitations for the meet up</li>
 +
<li>Organised speakers and planned the remainder of the meet up</li>
 +
<li>Continued research on assays and writing COSHH forms</li>
 +
</ul>
 +
</div>
</div>
-
 
+
</div>
-
<div class="pageSection2">
+
-
<div class="sectionHeading">
+
<div class="pageSection1">
-
<h2>Protocol 12: Sticky End ligation</h2>
+
<div class="sectionHeading">
-
</div>
+
<h2>Week 3</h2>
-
<div class="sectionContent">
+
-
<h3>Materials and Equipment</h3>
+
-
Eppendorf, p10, tips, reaction buffer, dH2O, restricted DNA, T4 Ligase.
+
-
<h3>Time</h3>
+
-
Prep: 5 minutes<br/>
+
-
Run: 30 minutes
+
-
<h3>Procedure</h3>
+
-
<ol>
+
-
<li>Make up eppendorfs containing the ligation mix, as follows:
+
-
<ul>
+
-
<li>2μ l reaction buffer.</li>
+
-
<li>10ng insert.</li>
+
-
<li>10ng plasmid.</li>
+
-
<li>1μ l T4 ligase.</li>
+
-
<li>Make up to 20μ l with dH2O.</li>
+
-
</ul>
+
-
</li>
+
-
<li>Flick to mix and leave for 30 minutes.</li>
+
-
</ol>
+
-
</div>
+
</div>
</div>
-
 
+
<div class="sectionContent">
-
<div class="pageSection1">
+
<h3>Monday 7th July</h3>
-
<div class="sectionHeading">
+
<ul>
-
<h2>Protocol 13: Restriction Digest</h2>
+
<li>Weekly meeting to set objectives</li>
-
</div>
+
<li>Write up policy and practices project outline</li>
-
<div class="sectionContent">
+
<li>Emailed Sheffield 2010 iGEM Team regarding their lab notation work</li>
-
<h3>Materials and Equipment</h3>
+
<li>Collated more contacts for sponsors</li>
-
Eppendorf, p10, p100, tips, restriction enzymes, ice, heat block.
+
<li>Contacted modelling expert within the University to set up a meeting</li>
-
<h3>Time</h3>
+
<li>Wiki development meeting</li>
-
Prep: 5 minutes<br/>
+
</ul>
-
Run: 1 hour
+
-
<h3>Procedure</h3>
+
<h3>Tuesday 8th July</h3>
-
<ol>
+
<ul>
-
<li>To each eppendorf add:
+
<li>Project implementation discussion and potential designs drawn on CAD</li>
-
<ol>
+
<li>Assay research</li>
-
<li>10 units of restriction enzyme 1 (usually 1μl).</li>
+
<li>Researched GMO approval requirements for the safety form</li>
-
<li>10 units of restriction enzyme 2 (usually 1μl).</li>
+
</ul>
-
<li>1μg DNA.</li>
+
-
<li>5μl 10x buffer.</li>
+
<h3>Wednesday 9th July</h3>
-
<li>Make up to 20μl with dH2O.</li>
+
<ul>
-
</ol>
+
<li>Wiki development</li>
-
</li>
+
<li>Sponsorship requests</li>
-
<li>Flick to mix.</li>
+
</ul>
-
<li>Incubate at 37°c for 60 minutes.</li>
+
-
<li>To stop the reaction, heat the sample to an appropriate temperature to inactivate the restriction enzyme used.</li>
+
<h3>Thursday 10th July</h3>
-
<li>Put on ice.</li>
+
<ul>
-
</ol>
+
<li>Sponsorship requests
-
</div>
+
<li>Arranged catering for the meet up</li>
 +
<li>Wiki development</li>
 +
<li>Discussed the improvement of the lab notation</li>
 +
<li>Assay database idea discussed</li>
 +
</ul>
 +
 +
<h3>Friday 11th July</h3>
 +
<ul>
 +
<li>Weekly meeting and report</li>
 +
<li>Reading research papers regarding FOGs and whey they are caused</li>
 +
<li>Lab notation development</li>
 +
<li>Collected assays for the database</li>
 +
<li>ERASynBio application sent </li>
 +
</ul>
 +
</div>
</div>
 +
</div>
 +
 +
<div class="pageSection2">
 +
<div class="sectionHeading">
 +
<h2>Week 4</h2>
 +
</div>
 +
<div class="sectionContent">
 +
<h3>Monday 14th July</h3>
 +
<ul>
 +
<li>Weekly meeting to set objectives</li>
 +
<li>Finalised logo design</li>
 +
<li>Made contact with experts at Northumbrian Water and Anglian Water</li>
 +
<li>Wiki development meeting</li>
 +
</ul>
 +
 +
<h3>Tuesday 15th July</h3>
 +
<ul>
 +
<li>Modelling meeting – use of Mathematica to analyse the FadR characterisation, use of CFD to model fluid flow and also control system analysis</li>
 +
<li>Meeting with industry expert from the University regarding product development</li>
 +
<li>Produced poster and presentation for the meet up on Friday</li>
 +
<li>Debugged temporary wiki</li>
 +
</ul>
 +
 +
<h3>Wednesday 16th July</h3>
 +
<ul>
 +
<li>Discussed second research question further; concluded it would focus on responsibility</li>
 +
<li>Jamboree meeting</li>
 +
</ul>
 +
 +
<h3>Thursday 17th July</h3>
 +
<ul>
 +
<li>Set up interviews with Sheffield City Council</li>
 +
<li>Sent reminder to all teams attending Friday’s meet up</li>
 +
<li>Finalised meet up details</li>
 +
</ul>
 +
 +
<h3>Friday 18th July</h3>
 +
<ul>
 +
<li>Meet up</li>
 +
</ul>
 +
 +
</div>
 +
</div>
 +
 +
<div class="pageSection1">
 +
<div class="sectionHeading">
 +
<h2>Week 5</h2>
 +
</div>
 +
<div class="sectionContent">
 +
<h3>Monday 21st July</h3>
 +
<ul>
 +
<li>Weekly meeting to set objectives</li>
 +
<li>Discussed interview questions for the responsibility question</li>
 +
<li>Modelling meeting – biological modelling may be limited; product design may be better to focus on</li>
 +
<li>FadR characterisation meeting – decided to continue characterising this but not use it in the final construct of our product</li>
 +
<li>CAD drawings of current designs</li>
 +
<li>Interview with University lecturer from Molecular Biology and Biotechnology</li>
 +
</ul>
 +
 +
<h3>Tuesday 22nd July</h3>
 +
<ul>
 +
<li>Researched current enzyme dosing systems to help develop product further</li>
 +
<li>Interview with a food outlet regarding responsibility</li>
 +
<li>Started transcribing lab book protocols into HTML code</li>
 +
<li>Researched the law and legislation regarding fat disposal by the public</li>
 +
</ul>
 +
 +
<h3>Wednesday 23rd July</h3>
 +
<ul>
 +
<li>Meeting with expert from Anglian Water</li>
 +
<li>Set up interviews within the University for the faculty wide study</li>
 +
<li>Skype meeting with Manchester iGEM Team for potential collaboration; found that projects were quite different</li>
 +
</ul>
 +
 +
<h3>Thursday 24th July</h3>
 +
<ul>
 +
<li>Used SolidWorks for new product designs</li>
 +
<li>Contacts schools regarding outreach</li>
 +
<li>Spoke to Oxford iGEM Team regarding collaboration</li>
 +
</ul>
 +
 +
<h3>Friday 25th July</h3>
 +
<ul>
 +
<li>Weekly meeting and report</li>
 +
<li>Decided to pursue the Manufacturing track</li>
 +
</ul>
 +
 +
</div>
 +
</div>
 +
 +
<div class="pageSection2">
 +
<div class="sectionHeading">
 +
<h2>Week 6</h2>
 +
</div>
 +
<div class="sectionContent">
 +
<h3>Monday 28th July</h3>
 +
<ul>
 +
<li>Weekly meeting to set objectives</li>
 +
<li>Reading FOG papers</li>
 +
<li>Began writing up parts of the project we have completed so far</li>
 +
<li>Collaborative links established with Valencia iGEM Team</li>
 +
</ul>
 +
 +
<h3>Tuesday 29th July</h3>
 +
<ul>
 +
<li>Interview with expert from Northumbrian Water</li>
 +
<li>Organised lab notation testing sessions and carried these out</li>
 +
<li>Collaborative links with Oxford iGEM  Team</li>
 +
<li>Interview with University lecturer in Landscape</li>
 +
</ul>
 +
 +
<h3>Wednesday 30th July</h3>
 +
<ul>
 +
<li>Analysed feedback from lab notation testing and made suggested improvements</li>
 +
<li>Interviews with University lecturers from the Medical School and Geography</li>
 +
<li>Began SocioBrick development and discussion</li>
 +
</ul>
 +
 +
<h3>Thursday 31st July</h3>
 +
<ul>
 +
<li>Contacted Paris Bettencourt iGEM Team to be featured in their newsletter</li>
 +
</ul>
 +
 +
<h3>Friday 2nd August</h3>
 +
<ul>
 +
<li>Weekly meeting and report</li>
 +
<li>Interviews with University lecturers from Civil Engineering and Biomedical Science</li>
 +
<li>SocioBrick development</li>
 +
</ul>
 +
 +
</div>
 +
</div>
 +
 +
<div class="pageSection1">
 +
<div class="sectionHeading">
 +
<h2>Week 7</h2>
 +
</div>
 +
<div class="sectionContent">
 +
<h3>Monday 4th August</h3>
 +
<ul>
 +
<li>Weekly meeting to set objectives</li>
 +
<li>Finished improved lab protocols</li>
 +
<li>Skype meetings with Oxford iGEM and Paris Bettencourt iGEM Teams </li>
 +
<li>Interview with a food outlet for the responsibility study</li>
 +
</ul>
 +
 +
<h3>Tuesday 5th August</h3>
 +
<ul>
 +
<li>Started writing up SocioBrick descriptions</li>
 +
<li>Wiki development</li>
 +
<li>Product design</li>
 +
</ul>
 +
 +
<h3>Wednesday 6th August</h3>
 +
<ul>
 +
<li>Interview with University lecturer from Automatic Control and Systems Engineering</li>
 +
<li>Wiki development</li>
 +
<li>Product design</li>
-
<div class="pageSection2">
+
</ul>
-
<div class="sectionHeading">
+
-
<h2>Protocol 14: Glycerol Stocks</h2>
+
<h3>Thursday 7th August</h3>
-
</div>
+
<ul>
-
<div class="sectionContent">
+
<li>Interviews with University lecturers from Music and Biblical Studies</li>
-
<i>Allows stocks of cells to be kept in the -80</i>
+
<li>Product design</li>
-
<h3>Materials and Equipment</h3>
+
<li>Analysis of interviews conducted over the past few weeks</li>
-
P1000 pipette, tips, eppendorf tubes, microwave, 100% glycerol, overnight cultures of cells.
+
 
-
<h3>Time</h3>
+
</ul>
-
Prep: 5 minutes<br/>
+
-
Run: 5 minutes
+
<h3>Friday 8th August</h3>
-
<h3>Procedure</h3>
+
<ul>
-
<ol>
+
<li>Weekly meeting and report</li>
-
<li>Take 800μl of overnight cells using a P1000 pipette and add into separate, labelled eppendorf tubes.</li>
+
<li>Skype meeting with Oxford iGEM Team</li>
-
<li>Microwave the 100% glycerol for 5 seconds.</li>
+
<li>Added to the wiki write ups</li>
-
<li>Take 200μl of the glycerol using a P1000 pipette and add into each eppendorf tube.</li>
+
</ul>
-
<li>Place on ice immediately.</li>
+
-
<li>Transfer to the -80°C freezer to be stored.</li>
+
</div>
-
</ol>
+
</div>
-
</div>
+
 +
<div class="pageSection2">
 +
<div class="sectionHeading">
 +
<h2>Week 8</h2>
 +
</div>
 +
<div class="sectionContent">
 +
<h3>Monday 11th August</h3>
 +
<ul>
 +
<li>Weekly meeting to set objectives</li>
 +
<li>Interview analysis</li>
 +
</ul>
 +
 +
<h3>Tuesday 12th August</h3>
 +
<ul>
 +
<li>Write up of the interviews conducted within the University</li>
 +
<li>Product development</li>
 +
<li>SocioBrick development</li>
 +
</ul>
 +
 +
<h3>Wednesday 13th August</h3>
 +
<ul>
 +
<li>Interview with a representative from the Labour party</li>
 +
<li>Wiki development</li>
 +
<li>Product development</li>
 +
</ul>
 +
 +
<h3>Thursday 14th August</h3>
 +
<ul>
 +
<li>SocioBrick development</li>
 +
<li>Product development</li>
 +
</ul>
 +
 +
<h3>Friday 15th August</h3>
 +
<ul>
 +
<li>Weekly meeting and report</li>
 +
<li>Wrote project abstract for the iGEM official team page</li>
 +
</ul>
 +
 +
</div>
 +
</div>
 +
 +
<div class="pageSection1">
 +
<div class="sectionHeading">
 +
<h2>Week 9</h2>
 +
</div>
 +
<div class="sectionContent">
 +
<h3>Monday 18th August</h3>
 +
<ul>
 +
<li>Weekly meeting to set objectives</li>
 +
<li>Researched more analysis SocioBricks as these were lacking</li>
 +
</ul>
 +
 +
<h3>Tuesday 19th August</h3>
 +
<ul>
 +
<li>Product design</li>
 +
<li>Interview analysis for responsibility report</li>
 +
<li>Wiki development</li>
 +
</ul>
 +
 +
<h3>Wednesday 20th August</h3>
 +
<ul>
 +
<li>Wiki team meeting to set up deadlines for the next few weeks</li>
 +
<li>Designs for the wiki layout made</li>
 +
<li>Writing up responsibility report</li>
 +
</ul>
 +
 +
<h3>Thursday 21st August</h3>
 +
<ul>
 +
<li>Writing up responsibility report</li>
 +
<li>Interviewed more food outlets and analysed the results</li>
 +
<li>Contacted more water companies</li>
 +
<li>Set up a trip to Veolia for late September </li>
 +
</ul>
 +
 +
<h3>Friday 22nd August</h3>
 +
<ul>
 +
<li>Weekly meeting and report</li>
 +
<li>Collaborative link with Valencia_UPV iGEM Team – agreed to test our lab notation</li>
 +
<li>Interview with a Labour councillor</li>
 +
</ul>
 +
 +
</div>
 +
</div>
 +
 +
<div class="pageSection2">
 +
<div class="sectionHeading">
 +
<h2>Week 10</h2>
 +
</div>
 +
<div class="sectionContent">
 +
<h3>Monday 25th August</h3>
 +
<ul>
 +
<li>Bank Holiday
 +
</ul>
 +
 +
<h3>Tuesday 26th August</h3>
 +
<ul>
 +
<li>Planned outreach events for schools</li>
 +
<li>Made poster for YSB/UCL meet up</li>
 +
<li>Sent improved lab notation to Edinburgh iGEM Team for collaboration</li>
 +
<li>Writing up responsibility report</li>
 +
</ul>
 +
 +
<h3>Wednesday 27th August</h3>
 +
<ul>
 +
<li>Made presentation for YSB/UCL meet up</li>
 +
<li>Product development</li>
 +
<li>Safety form</li>
 +
</ul>
 +
 +
<h3>Thursday 28th August</h3>
 +
<ul>
 +
<li>Treatment plant visit and interviews with the workers there</li>
 +
<li>Product development</li>
 +
</ul>
 +
 +
<h3>Friday 29th August</h3>
 +
<ul>
 +
<li>Weekly meeting and report – everyone given specific deadlines to meet over the next month</li>
 +
<li>Finalised poster and presentation for YSB/UCL meet up</li>
 +
<li>Wiki development</li>
 +
<li>Uploaded project title and abstract to the official iGEM team page</li> <li>Uploaded project title and abstract to the official iGEM team page</li>
 +
</ul>
 +
</div>
</div>
</div>
</div>
</body>
</body>
</html>
</html>

Revision as of 11:34, 17 October 2014

Dry Lab Journal

At the start of our project, we undertook a week of boot camp – yes, it was as intense as it sounds! With all team members coming from a range of backgrounds with different abilities, we thought that this would be a good idea to allow everyone to be on the same page with the same basic level of understanding in each component of the project.

Training Week

Monday 16th June

  • Laboratory Orientation and Safety Training
  • Laboratory Techniques 1
    • Pipetting, LB Broth, LB agar and Overnight Cultures
  • Project Expectations Discussion
    • Medal criteria, weekly meetings to discuss objectives and results, personal logs of what and how things have been done

Tuesday 17th June

  • Introduction of Policy and Practices
  • Discussion of Social Implications of Synthetic Biology
  • Genome 101
  • Laboratory Techniques 2
    • Plasmid DNA extraction, PCR and Chemical Transformation

Wednesday 18th June

  • Introduction to Modelling
  • BioBricks and the Registry
  • Methodology in Social Science and Data Collection

Thursday 19th June

  • Oxford Meet Up
  • Wiki and Team Identity Discussion

Friday 20th June

  • Project Presentation to the Chemical and Biological Engineering Department at the University of Sheffield
  • Weekly Report and Feedback

This week was very useful as it allowed us to become familiar with all aspects of the project. As well as this, it was a great icebreaker that helped our team of students, advisors and supervisors to get to know each other better!

Week 1

Monday 23rd June

  • Collated contact details of potential sponsors
  • Drafter sponsorship request letters for money, equipment and expertise

Tuesday 24th June

  • Continued working towards producing a sponsorship package
  • Reading published papers relating to synthetic biology and iGEM regarding awareness and impressions
  • Set up two meetings: marketing and industry expertise

Wednesday 25th June

  • Mathematica – 4 of our student team members went on a course to learn Mathematica for the modelling aspect of the project
  • Further reading of policy and practices papers
  • Worked on team identity in preparation for the marketing meeting
  • Sponsorship package completed – this can be found here
  • Translated written protocols into the lab notation

Thursday 26th June

  • Started planning our Meet Up and contacted teams for potential collaboration
  • Continued work on the lab notation
  • Discussed potential research questions to carry out a study within the University
  • Meeting with industry expert within the University of Sheffield; this provided us with new contacts and a better
    • understanding of the UK sewage system and how it functions. It was also very useful as we began to consider the applications of our project and what we may need to consider. Mathematica

Friday 27th June

  • Used XML and PYTHON to try to produce a computer language for the lab notation
  • Mathematica
  • Weekly meeting and report

Week 2

Monday 30th June

  • Weekly meeting to set objectives
  • Worked on the interview questions to be used for the University of Sheffield case study
  • Further research on potential sponsors
  • Mathematica

Tuesday 1st July

  • Meeting with advisors to discuss suitability of research question and interviews
  • Began to consider the ethics needed to go alongside this
  • Mathematica

Wednesday 2nd July

  • Finalised research questions and sent off ethics approval
  • Began learning HTML and other mark up languages for the Wiki

Thursday 3rd July

  • Team identity discussion on potential project names and logo
  • Researched assays
  • Wrote COSHH forms
  • Sent out sponsorship packages
  • ERASynBio research and application draft

Friday 4th July

  • Sent out invitations for the meet up
  • Organised speakers and planned the remainder of the meet up
  • Continued research on assays and writing COSHH forms

Week 3

Monday 7th July

  • Weekly meeting to set objectives
  • Write up policy and practices project outline
  • Emailed Sheffield 2010 iGEM Team regarding their lab notation work
  • Collated more contacts for sponsors
  • Contacted modelling expert within the University to set up a meeting
  • Wiki development meeting

Tuesday 8th July

  • Project implementation discussion and potential designs drawn on CAD
  • Assay research
  • Researched GMO approval requirements for the safety form

Wednesday 9th July

  • Wiki development
  • Sponsorship requests

Thursday 10th July

  • Sponsorship requests
  • Arranged catering for the meet up
  • Wiki development
  • Discussed the improvement of the lab notation
  • Assay database idea discussed

Friday 11th July

  • Weekly meeting and report
  • Reading research papers regarding FOGs and whey they are caused
  • Lab notation development
  • Collected assays for the database
  • ERASynBio application sent

Week 4

Monday 14th July

  • Weekly meeting to set objectives
  • Finalised logo design
  • Made contact with experts at Northumbrian Water and Anglian Water
  • Wiki development meeting

Tuesday 15th July

  • Modelling meeting – use of Mathematica to analyse the FadR characterisation, use of CFD to model fluid flow and also control system analysis
  • Meeting with industry expert from the University regarding product development
  • Produced poster and presentation for the meet up on Friday
  • Debugged temporary wiki

Wednesday 16th July

  • Discussed second research question further; concluded it would focus on responsibility
  • Jamboree meeting

Thursday 17th July

  • Set up interviews with Sheffield City Council
  • Sent reminder to all teams attending Friday’s meet up
  • Finalised meet up details

Friday 18th July

  • Meet up

Week 5

Monday 21st July

  • Weekly meeting to set objectives
  • Discussed interview questions for the responsibility question
  • Modelling meeting – biological modelling may be limited; product design may be better to focus on
  • FadR characterisation meeting – decided to continue characterising this but not use it in the final construct of our product
  • CAD drawings of current designs
  • Interview with University lecturer from Molecular Biology and Biotechnology

Tuesday 22nd July

  • Researched current enzyme dosing systems to help develop product further
  • Interview with a food outlet regarding responsibility
  • Started transcribing lab book protocols into HTML code
  • Researched the law and legislation regarding fat disposal by the public

Wednesday 23rd July

  • Meeting with expert from Anglian Water
  • Set up interviews within the University for the faculty wide study
  • Skype meeting with Manchester iGEM Team for potential collaboration; found that projects were quite different

Thursday 24th July

  • Used SolidWorks for new product designs
  • Contacts schools regarding outreach
  • Spoke to Oxford iGEM Team regarding collaboration

Friday 25th July

  • Weekly meeting and report
  • Decided to pursue the Manufacturing track

Week 6

Monday 28th July

  • Weekly meeting to set objectives
  • Reading FOG papers
  • Began writing up parts of the project we have completed so far
  • Collaborative links established with Valencia iGEM Team

Tuesday 29th July

  • Interview with expert from Northumbrian Water
  • Organised lab notation testing sessions and carried these out
  • Collaborative links with Oxford iGEM Team
  • Interview with University lecturer in Landscape

Wednesday 30th July

  • Analysed feedback from lab notation testing and made suggested improvements
  • Interviews with University lecturers from the Medical School and Geography
  • Began SocioBrick development and discussion

Thursday 31st July

  • Contacted Paris Bettencourt iGEM Team to be featured in their newsletter

Friday 2nd August

  • Weekly meeting and report
  • Interviews with University lecturers from Civil Engineering and Biomedical Science
  • SocioBrick development

Week 7

Monday 4th August

  • Weekly meeting to set objectives
  • Finished improved lab protocols
  • Skype meetings with Oxford iGEM and Paris Bettencourt iGEM Teams
  • Interview with a food outlet for the responsibility study

Tuesday 5th August

  • Started writing up SocioBrick descriptions
  • Wiki development
  • Product design

Wednesday 6th August

  • Interview with University lecturer from Automatic Control and Systems Engineering
  • Wiki development
  • Product design

Thursday 7th August

  • Interviews with University lecturers from Music and Biblical Studies
  • Product design
  • Analysis of interviews conducted over the past few weeks

Friday 8th August

  • Weekly meeting and report
  • Skype meeting with Oxford iGEM Team
  • Added to the wiki write ups

Week 8

Monday 11th August

  • Weekly meeting to set objectives
  • Interview analysis

Tuesday 12th August

  • Write up of the interviews conducted within the University
  • Product development
  • SocioBrick development

Wednesday 13th August

  • Interview with a representative from the Labour party
  • Wiki development
  • Product development

Thursday 14th August

  • SocioBrick development
  • Product development

Friday 15th August

  • Weekly meeting and report
  • Wrote project abstract for the iGEM official team page

Week 9

Monday 18th August

  • Weekly meeting to set objectives
  • Researched more analysis SocioBricks as these were lacking

Tuesday 19th August

  • Product design
  • Interview analysis for responsibility report
  • Wiki development

Wednesday 20th August

  • Wiki team meeting to set up deadlines for the next few weeks
  • Designs for the wiki layout made
  • Writing up responsibility report

Thursday 21st August

  • Writing up responsibility report
  • Interviewed more food outlets and analysed the results
  • Contacted more water companies
  • Set up a trip to Veolia for late September

Friday 22nd August

  • Weekly meeting and report
  • Collaborative link with Valencia_UPV iGEM Team – agreed to test our lab notation
  • Interview with a Labour councillor

Week 10

Monday 25th August

  • Bank Holiday

Tuesday 26th August

  • Planned outreach events for schools
  • Made poster for YSB/UCL meet up
  • Sent improved lab notation to Edinburgh iGEM Team for collaboration
  • Writing up responsibility report

Wednesday 27th August

  • Made presentation for YSB/UCL meet up
  • Product development
  • Safety form

Thursday 28th August

  • Treatment plant visit and interviews with the workers there
  • Product development

Friday 29th August

  • Weekly meeting and report – everyone given specific deadlines to meet over the next month
  • Finalised poster and presentation for YSB/UCL meet up
  • Wiki development
  • Uploaded project title and abstract to the official iGEM team page
  • Uploaded project title and abstract to the official iGEM team page

Retrieved from "http://2014.igem.org/Template:Team:Sheffield/Content:LabProtocols"