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| | <!-- THIS IS WHERE THE CONTENT GOES--> | | <!-- THIS IS WHERE THE CONTENT GOES--> |
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| | <div class="col-md-9 content-area"> | | <div class="col-md-9 content-area"> |
| | <div class="content-container"> | | <div class="content-container"> |
| | <h1>Our Notebook</h1> | | <h1>Our Notebook</h1> |
| | <p> | | <p> |
| − | <h3>July 20th, 2016</h3> | + | <h3><a href="https://static.igem.org/mediawiki/2016/b/b8/T--UrbanTundra_Edmonton--JuneNotebook_.pdf" target="_blank">June Lab Notebook</a></h3> |
| − | <ol>
| + | <h3><a href="https://static.igem.org/mediawiki/2016/5/5e/T--UrbanTundra_Edmonton--JulyNotebook_.pdf" target="_blank">July Lab Notebook</a></h3> |
| − | <li>4 microlitres of milliq water with 1 microlitre of plasmid</li>
| + | <h3><a href="https://static.igem.org/mediawiki/2016/5/5d/T--UrbanTundra_Edmonton--AugustNotebook_.pdf" target="_blank">August Lab Notebook</a></h3> |
| − | <li>5 microlitres of no chlorite dismutase</li>
| + | <h3><a href="https://static.igem.org/mediawiki/2016/e/eb/T--UrbanTundra_Edmonton--SeptemberNotebook_.pdf" target="_blank">September Lab Notebook</a></h3> |
| − | <li>1 microlitre of cutsmart buffer (specifically 1.1111 microlitre)</li>
| + | |
| − | <ul>A. 10 x cutsmart buffer into tubes makes 1x [ ]</ul>
| + | |
| − | <li>When adding BsaI, only touch the surface of the BsaI to the pipette tip. The reason: has glycerol which is sticky. Take 0.5 microlitre of BsaI.</li>
| + | |
| − | <li>Mix sample very gently then put into a 37 degrees celsius incubator for 1 hour. </li>
| + | |
| − | <li>Heat kill at 65 degrees celsius for 20 minutes. This kills the BsaI to prevent star activity. </li>
| + | |
| − | <li>Pulse down tubes then add 8 microlitre of milliq water. </li>
| + | |
| − | <li>Add 2 microlitres of T4DNA ligase buffer. </li>
| + | |
| − | <li>Add 0.5 microlitre of ligase then mix gently and incubate at room temperature for 30 minutes or longer.
| + | |
| − | </li>
| + | |
| − | </ol>
| + | |
| | | | |
| − | <br><hr></hr></br>
| |
| − |
| |
| − | <h3>July 21st, 2016</h3>
| |
| − | <ol>
| |
| − | <li>Did some plating. </li>
| |
| − | <li>Plated three agar types:</li>
| |
| − | <ul>A. 4 plates of no antibiotic </ul>
| |
| − | <ul>B. 10 plates of kanamycin (CM)</ul>
| |
| − | <ul>C. 10 plates of ampicillin (Amp)</ul>
| |
| − | <li>Purpose: try to find the antibiotic marker on bacteria.</li>
| |
| − | <li>Observational expectations: If it’s purple, then there is no Cld gene sequence. If white then has CLd gene sequence. </li>
| |
| − | <li>Lives or dies on certain plates. Alive on ampicillin, then it has the marker. Dies it has the CM marker. </li>
| |
| − | <li>Added 20 microlitres in each plate</li>
| |
| − | <ul>A. A total of 7 plates</ul>
| |
| − | <ul>B. DNA minus has none, CM, and AMP</ul>
| |
| − | <ul>C. Positive control has CM and AMP</ul>
| |
| − | <ul>D. Cld +/- has CM and AMP</ul>
| |
| | </ol> | | </ol> |
| − |
| |
| − | <h3>Competent cell transformation:</h3>
| |
| − | <ol>
| |
| − | <li>1 microlitre of positive control added to competent cells (positive control: CPB we used yesterday before cutting and gluing)</li>
| |
| − | <li>10 microlitre of cloned plasmid added to competent cells (Cloned plasmid is what we did yesterday).
| |
| − | </li>
| |
| − | <li>Positive control, negative control, and cloned plasmid were chilled for 30 minutes, heat shocked for 90 seconds, chilled on ice for 5 minutes, and finally incubated at 37 degrees celsius with 1 ml of LB broth.</li>
| |
| − | </ol>
| |
| − |
| |
| − | <br><hr></hr></br>
| |
| − |
| |
| − | <h3>July 22nd, 2016</h3>
| |
| − | <ol>
| |
| − | <li>It was observed that there were little purple colonies, however the colors will develope more over a longer period.</li>
| |
| − | <li>Results from yesterday:</li>
| |
| − | <ul>A. No Ab plates: very thick growth</ul>
| |
| − | <ul>B. Amp plates: no growth: conclusion: no amp marker</ul>
| |
| − | <ul>C. CM: growth</ul>
| |
| − | <li>Results chart:</li>
| |
| − | <table style="width:96%">
| |
| − | <tr>
| |
| − | <th></th>
| |
| − | <th>+ control</th>
| |
| − | <th>DNA -</th>
| |
| − | <th>Cld +</th>
| |
| − | <th>Cld -</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>No Antibiotic</td>
| |
| − | <td></td>
| |
| − | <td>Yes, very thick</td>
| |
| − | <td></td>
| |
| − | <td></rd>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Amp</td>
| |
| − | <td>No growth</td>
| |
| − | <td>No growth</td>
| |
| − | <td>No growth</td>
| |
| − | <td>No growth</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>CM</td>
| |
| − | <td>Growth (purple colonies)</td>
| |
| − | <td>No growth</td>
| |
| − | <td>Possibly purple and white*</td>
| |
| − | <td>Possible purple and white*</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | <p>* Purple colour will develop more over larger period of time (perhaps over the weekend)<p>
| |
| − | </ol>
| |
| − |
| |
| − | <br><hr></hr></br>
| |
| − |
| |
| − | <h3>July 23rd, 2016</h3>
| |
| − | <ol>
| |
| − | <p>Today was spent trying to get sponsors.</p>
| |
| − | </ol>
| |
| − |
| |
| − | <br><hr></hr></br>
| |
| − |
| |
| − | <h3>July 25th, 2016</h3>
| |
| − | <ol>
| |
| − | <p>Lab work: Today we created overnight cultures through inoculation for miniprep tomorrow</p>
| |
| − | <li>Pipette 5 ml of LB broth into 6 tubes</li>
| |
| − | <li>Add CM for antibiotic in the Broth</li>
| |
| − | <ul>A. Had to do the above procedure again</ul>
| |
| − | <li>Have 60 ml of LB broth</li>
| |
| − | <ul>A. One tube gets 50 ml → 50 microlitres of CM. </ul>
| |
| − | <li>Put 5 ml into culture tube</li>
| |
| − | <li>Inoculate bacteria grown from last lab day.</li>
| |
| − | <p>-CM allows only bacterias with CM markers to exist, thus allowing only organisms with the designated sequence to survive and live.</p>
| |
| − | </ol>
| |
| − | <p>After that, more fundraising work was done.</p>
| |
| − |
| |
| − | <br><hr></hr></br>
| |
| − |
| |
| − | <h3>July 26th, 2016</h3>
| |
| − | <ol>
| |
| − | <li>Yesterday’s labwork had to be redone since the inoculation wasn’t done correctly.</li>
| |
| − | <li>Reason: pipette tips used were too short and because the top of the tips were contaminated, the whole culture became contaminated.</li>
| |
| − | <li>In our redone experiment, we used inoculation stick instead (‘just to play it safe’ Mike reasoned)</li>
| |
| − | </ol>
| |
| − |
| |
| − | <br><hr></hr></br>
| |
| − |
| |
| − | <h3>July 27th, 2016</h3>
| |
| − | <ol>
| |
| − | <p><b>Miniprep day</b></p>
| |
| − | <p>Cld+ cultures from yesterday were purple- that’s not good (we want white ones) so we re-inoculated Cld+ for another miniprep tomorrow</p>
| |
| − | <p>Cld- cultures from yesterday were good so we are able to perform miniprep today with the Cld-</p>
| |
| − | <li>Microcentrifuge 1 ml of culture (do this three times to allow a large amount of cells to build up)</li>
| |
| − | <ul>A. These cells should be orange-reddish in colour. The reason for this is due to the production of a ‘heme’ group- that is a protein that binds with Fe.</ul>
| |
| − | <li>Addition of 250 microlitres of Buffer-PI allows resuspension of bacteria.</li>
| |
| − | <ul>A. Buffer PI was spilled on the table, but another bottle was brought down.</ul>
| |
| − | <ul>B. To thoroughly mix it, vortex the microcentrifuge column.</ul>
| |
| − | <li>Add 250 microlitres of Buffer P2. Mix by inverting it 4-6 times.</li>
| |
| − | <ul>A. Don’t let this sit for more than 5 minutes.</ul>
| |
| − | <li>Add 350 microlitres of Buffer N3 and mix by inverting 4-6 times.</li>
| |
| − | <ul>A. A whitish precipitate should have formed.</ul>
| |
| − | <li>Microcentrifuge for 10 minutes at >10 000 rpm.</li>
| |
| − | <ul>A. Supernatant is what we want. The solid is the garbage.</ul>
| |
| − | <ul>B. Once taken out, supernatant will be pipetted out into a separate tube (not a spin column). This allows unison of the supernatant and consistency</ul>
| |
| − | <ul>C. *Note* Eppendorf tubes are microcentrifuge tubes</ul>
| |
| − | <li>Pipette out supernatant from new microcentrifuge into labeled spin columns</li>
| |
| − | <ul>A. Centrifuge it for 30-60 seconds</ul>
| |
| − | <ul>B. Let it sit for a bit</ul>
| |
| − | <li>Add 0.75 ml of Buffer PE and centrifuge for 30-60s.</li>
| |
| − | <ul>A. Discard flow-through and centrifuge one more time for 1 minute.</ul>
| |
| − | <ul>B. Residual wash buffer (remove it)</ul>
| |
| − | <li>Add 100 microlitres of Buffer EB after transferring spin column over a new microcentrifuge tube.</li>
| |
| − | <ul>A. Stand for one minute</ul>
| |
| − | <ul>B. Spin for one minute</ul>
| |
| − | <ul>C. *Technique* Using finger to balance it and place it just above the filtre- Don’t touch the filter.
| |
| − | </ul>
| |
| − | <li>Now, we will test concentration and purity through the use of a spectrophotometer.</li>
| |
| − | <li>Results:</li>
| |
| − | <table style="width:96%">
| |
| − | <tr>
| |
| − | <th></th>
| |
| − | <th>Vial Number</th>
| |
| − | <th>260/280</th>
| |
| − | <th>Concentration in nanograms/microlitre</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>1</td>
| |
| − | <td>1.77</td>
| |
| − | <td>88.4</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>1</td>
| |
| − | <td>1.80</td>
| |
| − | <td>85.0</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>2</td>
| |
| − | <td>1.81</td>
| |
| − | <td>84.4</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>2</td>
| |
| − | <td>1.80</td>
| |
| − | <td>85.0</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>3</td>
| |
| − | <td>1.79</td>
| |
| − | <td>87.0</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>3</td>
| |
| − | <td>1.81</td>
| |
| − | <td>78.6</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>4</td>
| |
| − | <td>1.79</td>
| |
| − | <td>83.6</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>4</td>
| |
| − | <td>1.79</td>
| |
| − | <td>83.1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>5</td>
| |
| − | <td>1.79</td>
| |
| − | <td>77.7</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>5</td>
| |
| − | <td>1.77</td>
| |
| − | <td>81.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>6</td>
| |
| − | <td>1.81</td>
| |
| − | <td>76.7</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>6</td>
| |
| − | <td>1.79</td>
| |
| − | <td>77.2</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </ol>
| |
| − |
| |
| − | <br><hr></hr></br>
| |
| − |
| |
| − | <h3>July 28th 2016</h3>
| |
| − | <ol>
| |
| − | <p><b>Miniprep (again) today</b></p>
| |
| − | <li>Cld + cultures are ready (not purple this time)</li>
| |
| − | <ul>A. Perform miniprep on them</ul>
| |
| − | <ul>B. We went through the exact procedure as the previous day</ul>
| |
| − | </ol>
| |
| − |
| |
| − | <h3>Miniprep</h3>
| |
| − | <ol>
| |
| − | <li>Observations: Like yesterday, the pellet that formed after the microcentrifugation is reddish-orange</li>
| |
| − | <ul>A. Vial 4 (after vortex with Buffer PI) appears to be purplish in colour rather than pink like the rest.</ul>
| |
| − | <ul>B. Pink: “Strawberry Milkshake”</ul>
| |
| − | <ul>C. Purple: “Glove coloured purple with a slight tint of pink”</ul>
| |
| − | <li>Second centrifugation (with PE) of number 4: the waste was blue and this might be due to the blue dye of the sharpie we used to differentiate vial 4’s content from the rest.</li>
| |
| − | <ul>A. At first, it was believed this was due to the initial purple colour, but upon closer inspection, it was realized some blue streaked marks suggested that enthanol cleaned away the blue sharpie dye.</ul>
| |
| − | <li>Results:</li>
| |
| − | <table style="width:96%">
| |
| − | <tr>
| |
| − | <th></th>
| |
| − | <th>Vial Number</th>
| |
| − | <th>260/280</th>
| |
| − | <th>Concentration in nanograms/microlitre</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>1</td>
| |
| − | <td>1.78</td>
| |
| − | <td>86.7</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>1</td>
| |
| − | <td>1.83</td>
| |
| − | <td>85.4</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>2</td>
| |
| − | <td>1.79</td>
| |
| − | <td>98.5</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>2</td>
| |
| − | <td>1.79</td>
| |
| − | <td>98.6</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>3</td>
| |
| − | <td>1.83</td>
| |
| − | <td>81.9</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>3</td>
| |
| − | <td>N/A</td>
| |
| − | <td>N/A</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 3</td>
| |
| − | <td>3</td>
| |
| − | <td>1.82</td>
| |
| − | <td>82.9</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>4</td>
| |
| − | <td>1.82</td>
| |
| − | <td>97.1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>4</td>
| |
| − | <td>1.80</td>
| |
| − | <td>97.3</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>5</td>
| |
| − | <td>1.82</td>
| |
| − | <td>92.7</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>5</td>
| |
| − | <td>1.82</td>
| |
| − | <td>93.9</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>6</td>
| |
| − | <td>1.82</td>
| |
| − | <td>95.4</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>6</td>
| |
| − | <td>1.82</td>
| |
| − | <td>95.4</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | <ul>A. *N/A is due to pipette contamination. The 260/280 value here was 1.64 and the concentration reading was 93.8 nanograms/microlitre</ul>
| |
| − | <ul>B. Cld+ may have higher concentrations due to histidines present in the Cld+ sequence.</ul>
| |
| − | <li>Next, we created a recipe for mixmaster:</li>
| |
| − | <ul>A. It was calculated through the formula C1V1=C2V2 that the it was required 26.666… or 27 microlitres of cutsmart. 171 microlitres of milliculewater and 2 microlitres of BsaI.</ul>
| |
| − | <li>Gel Electrophoresis</li>
| |
| − | <ul>A. To check the plasmid backbone and the Cld +/- insert. </ul>
| |
| − | <ul>B. Separate (and no gluing)</ul>
| |
| − | <li>Create master mix by adding 27 microlitres of cutsmart, 171 microlitres of millicule water and 2 microlitres of BsaI. *remember that enzymes are always kept on ice!*</li>
| |
| − | <li>Transfer fifteen microlitres of master mix into each digestion tube (a total of 13 tubes)</li>
| |
| − | <ul>A. Each tube is labeled with a digit from 1 to 6 and whether it was Cld - or +. </ul>
| |
| − | <li>Add 5 microlitres of DNA to each tube.</li>
| |
| − | <ul>A. *note: cutsmart buffer is used to provide optimal environment for cut enzyme.</ul>
| |
| − | <ul>B. Cut DNA to compare relative sizes: we will see:</ul>
| |
| − | <ul>a. Cld +/- (1 Kb) </ul>
| |
| − | <ul>b. Colour gene (0.7 Kb)</ul>
| |
| − | <ul>c. Whole plasmid backbone (4 Kb)</ul>
| |
| − | <b>~Lunch break~</b>
| |
| − | <li>Gel electrophoresis</li>
| |
| − | <ul>A. Make gel: 1% agarose in TAE into well</ul>
| |
| − | <ul>a. *1% is 1g/100ml</ul>
| |
| − | <ul>B. Add loading dye into tubes</ul>
| |
| − | <ul>C. Add 5 microlitres of sample into wells</ul>
| |
| − | <ul>D. Same procedure as practice procedures found online</ul>
| |
| − | <li>Pcitures of results are taken....</li>
| |
| − | </ol>
| |
| − |
| |
| − | <br><hr></hr></br>
| |
| − |
| |
| − | <h3>August 2nd, 2016</h3>
| |
| − | <ol>
| |
| − | <li>We split the team up into non-science and science groups...</li>
| |
| − | </ol>
| |
| − |
| |
| − | <br><hr></hr></br>
| |
| − |
| |
| − | <h3>August 3rd, 2016</h3>
| |
| − | <ol>
| |
| − | <ul>
| |
| − | <li>Science based</li>
| |
| − | <ul>
| |
| − | <li>Perchlorate team initiates</li>
| |
| − | <li>Biobrick team initiates</li>
| |
| − | <li>Process and quant bio are to design the experiments and must research up on it. (This requires some time)</li>
| |
| − | </ul>
| |
| − | <li>Non science based</li>
| |
| − | <ul>
| |
| − | <li>Fundraising work was done</li>
| |
| − | </ul>
| |
| − | </ul>
| |
| − | <ul>
| |
| − | <li>Biobrick team: tomorrow, they will perform miniprepe after inoculating bacteria replicated today.</li>
| |
| − | <ul>
| |
| − | <li>Today, chemical transformation was performed (refer to 8.5) to create a lot of plasmids (there is not enough)</li>
| |
| − | <ul>
| |
| − | <ul>
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| − | <li>*When referring, note that ligase was not added</li>
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| − | </ul>
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| − | <li>Plasmid that were used were those given from igem (required in order to win gold).</li>
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| − | <li>Today, inoculation of grown bacteria will be performed to create cultures for miniprep tomorrow.</li>
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| − | </ul>
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| − | <li>Chloramphenicol: antibiotic will be added to the inoculated cultures.</li>
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| − | <ul>
| |
| − | <li>The given plasmids have a chloramphenicol resistance marker.</li>
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| − | </ul>
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| − | <li>All in all: the purpose of today for the biobrick team: Create more plasmids because there isn’t enough.</li>
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| − | <ul>
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| − | <li>Then. create cultures for miniprep.</li>
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| − | </ul>
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| − | </ul>
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| − | </ul>
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| − | <ul>
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| − | <li><b>Procedure of the Biobrick team:</b></li>
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| − | </ul>
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| − | <ol>
| |
| − | <li>Take out chemically competent e.coli from freezer and put in ice. 50 microlitres of e.coli into microcentrifuge tube.</li>
| |
| − | <li>DNA are added to cells. Stir with pipette tip, but do not pipette up or down.</li>
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| − | <li>Incubate on ice for 30 minutes.</li>
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| − | <li>Heat shock at 42 degrees celsius for 30 seconds.</li>
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| − | <li>Incubate for 5 minutes on ice, then add 1 ml of LB.</li>
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| − | <li>Incubate for 1 hr at 37 degrees celsius at 250 rpm. (5ml of LB and 5 microlitres of Antibiotic)</li>
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| − | <li>2 culture tubes: 5 ml of LB, 15 microlitres of antibiotic, and 100 microlitres of plasmid.</li>
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| − | <li>Incubate for whole night.</li>
| |
| − | </ol>
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| − |
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| − | <br><hr></hr></br>
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| − |
| |
| − | <p><strong> </strong></p>
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| − | <p><strong> August 4th, 2016</strong></p>
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| − | <ul>
| |
| − | <li>Inoculation from yesterday failed</li>
| |
| − | <li>Therefore, we must do it again</li>
| |
| − | <ul>
| |
| − | <li>Two tubes: one of each has: 10 microlitres of plasmid and 5 microlitres of antibiotic</li>
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| − | </ul>
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| − | </ul>
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| − | <ol>
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| − | <li>10 microlitres of plasmid into 1 microcentrifuge tube</li>
| |
| − | <li>Incubate on ice for 30 minutes</li>
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| − | <li>Heat shock at 42 degrees celsius for 90 seconds</li>
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| − | <li>Incubate on ice for 5 minutes</li>
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| − | <li>Add 1 ml of LB broth into tube and incubate at 37 degrees celsius for 1 hour.</li>
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| − | <li>Add 5 ml of LB broth into 2 culture tubes</li>
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| − | <li>Add 5 microlitres of antibiotic into each culture tubes</li>
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| − | <li>Transfer 200 microlitres of plasmid and e.coli into each culture tubes (Plasmid is called pSBIC3)
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| − | </li>
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| − | <li>Shake overnight at 37 degrees celsius. </li>
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| − | </ol>
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| − | <div class="column full_size">
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| − |
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| − | <h3>July 20th, 2016</h3>
| |
| − | <ol>
| |
| − | <li>4 microlitres of milliq water with 1 microlitre of plasmid</li>
| |
| − | <li>5 microlitres of no chlorite dismutase</li>
| |
| − | <li>1 microlitre of cutsmart buffer (specifically 1.1111 microlitre)</li>
| |
| − | <ul>A. 10 x cutsmart buffer into tubes makes 1x [ ]</ul>
| |
| − | <li>When adding BsaI, only touch the surface of the BsaI to the pipette tip. The reason: has glycerol which is sticky. Take 0.5 microlitre of BsaI.</li>
| |
| − | <li>Mix sample very gently then put into a 37 degrees celsius incubator for 1 hour. </li>
| |
| − | <li>Heat kill at 65 degrees celsius for 20 minutes. This kills the BsaI to prevent star activity. </li>
| |
| − | <li>Pulse down tubes then add 8 microlitre of milliq water. </li>
| |
| − | <li>Add 2 microlitres of T4DNA ligase buffer. </li>
| |
| − | <li>Add 0.5 microlitre of ligase then mix gently and incubate at room temperature for 30 minutes or longer.
| |
| − | </li>
| |
| − | </ol>
| |
| − |
| |
| − | <br><hr></hr></br>
| |
| − |
| |
| − | <h3>July 21st, 2016</h3>
| |
| − | <ol>
| |
| − | <li>Did some plating. </li>
| |
| − | <li>Plated three agar types:</li>
| |
| − | <ul>A. 4 plates of no antibiotic </ul>
| |
| − | <ul>B. 10 plates of kanamycin (CM)</ul>
| |
| − | <ul>C. 10 plates of ampicillin (Amp)</ul>
| |
| − | <li>Purpose: try to find the antibiotic marker on bacteria.</li>
| |
| − | <li>Observational expectations: If it’s purple, then there is no Cld gene sequence. If white then has CLd gene sequence. </li>
| |
| − | <li>Lives or dies on certain plates. Alive on ampicillin, then it has the marker. Dies it has the CM marker. </li>
| |
| − | <li>Added 20 microlitres in each plate</li>
| |
| − | <ul>A. A total of 7 plates</ul>
| |
| − | <ul>B. DNA minus has none, CM, and AMP</ul>
| |
| − | <ul>C. Positive control has CM and AMP</ul>
| |
| − | <ul>D. Cld +/- has CM and AMP</ul>
| |
| − | </ol>
| |
| − |
| |
| − | <h3>Competent cell transformation:</h3>
| |
| − | <ol>
| |
| − | <li>1 microlitre of positive control added to competent cells (positive control: CPB we used yesterday before cutting and gluing)</li>
| |
| − | <li>10 microlitre of cloned plasmid added to competent cells (Cloned plasmid is what we did yesterday).
| |
| − | </li>
| |
| − | <li>Positive control, negative control, and cloned plasmid were chilled for 30 minutes, heat shocked for 90 seconds, chilled on ice for 5 minutes, and finally incubated at 37 degrees celsius with 1 ml of LB broth.</li>
| |
| − | </ol>
| |
| − |
| |
| − | <br><hr></hr></br>
| |
| − |
| |
| − | <h3>July 22nd, 2016</h3>
| |
| − | <ol>
| |
| − | <li>It was observed that there were little purple colonies, however the colors will develope more over a longer period.</li>
| |
| − | <li>Results from yesterday:</li>
| |
| − | <ul>A. No Ab plates: very thick growth</ul>
| |
| − | <ul>B. Amp plates: no growth: conclusion: no amp marker</ul>
| |
| − | <ul>C. CM: growth</ul>
| |
| − | <li>Results chart:</li>
| |
| − | <table style="width:96%">
| |
| − | <tr>
| |
| − | <th></th>
| |
| − | <th>+ control</th>
| |
| − | <th>DNA -</th>
| |
| − | <th>Cld +</th>
| |
| − | <th>Cld -</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>No Antibiotic</td>
| |
| − | <td></td>
| |
| − | <td>Yes, very thick</td>
| |
| − | <td></td>
| |
| − | <td></rd>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Amp</td>
| |
| − | <td>No growth</td>
| |
| − | <td>No growth</td>
| |
| − | <td>No growth</td>
| |
| − | <td>No growth</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>CM</td>
| |
| − | <td>Growth (purple colonies)</td>
| |
| − | <td>No growth</td>
| |
| − | <td>Possibly purple and white*</td>
| |
| − | <td>Possible purple and white*</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | <p>* Purple colour will develop more over larger period of time (perhaps over the weekend)<p>
| |
| − | </ol>
| |
| − |
| |
| − | <br><hr></hr></br>
| |
| − |
| |
| − | <h3>July 23rd, 2016</h3>
| |
| − | <ol>
| |
| − | <p>Today was spent trying to get sponsors.</p>
| |
| − | </ol>
| |
| − |
| |
| − | <br><hr></hr></br>
| |
| − |
| |
| − | <h3>July 25th, 2016</h3>
| |
| − | <ol>
| |
| − | <p>Lab work: Today we created overnight cultures through inoculation for miniprep tomorrow</p>
| |
| − | <li>Pipette 5 ml of LB broth into 6 tubes</li>
| |
| − | <li>Add CM for antibiotic in the Broth</li>
| |
| − | <ul>A. Had to do the above procedure again</ul>
| |
| − | <li>Have 60 ml of LB broth</li>
| |
| − | <ul>A. One tube gets 50 ml → 50 microlitres of CM. </ul>
| |
| − | <li>Put 5 ml into culture tube</li>
| |
| − | <li>Inoculate bacteria grown from last lab day.</li>
| |
| − | <p>-CM allows only bacterias with CM markers to exist, thus allowing only organisms with the designated sequence to survive and live.</p>
| |
| − | </ol>
| |
| − | <p>After that, more fundraising work was done.</p>
| |
| − |
| |
| − | <br><hr></hr></br>
| |
| − |
| |
| − | <h3>July 26th, 2016</h3>
| |
| − | <ol>
| |
| − | <li>Yesterday’s labwork had to be redone since the inoculation wasn’t done correctly.</li>
| |
| − | <li>Reason: pipette tips used were too short and because the top of the tips were contaminated, the whole culture became contaminated.</li>
| |
| − | <li>In our redone experiment, we used inoculation stick instead (‘just to play it safe’ Mike reasoned)</li>
| |
| − | </ol>
| |
| − |
| |
| − | <br><hr></hr></br>
| |
| − |
| |
| − | <h3>July 27th, 2016</h3>
| |
| − | <ol>
| |
| − | <p><b>Miniprep day</b></p>
| |
| − | <p>Cld+ cultures from yesterday were purple- that’s not good (we want white ones) so we re-inoculated Cld+ for another miniprep tomorrow</p>
| |
| − | <p>Cld- cultures from yesterday were good so we are able to perform miniprep today with the Cld-</p>
| |
| − | <li>Microcentrifuge 1 ml of culture (do this three times to allow a large amount of cells to build up)</li>
| |
| − | <ul>A. These cells should be orange-reddish in colour. The reason for this is due to the production of a ‘heme’ group- that is a protein that binds with Fe.</ul>
| |
| − | <li>Addition of 250 microlitres of Buffer-PI allows resuspension of bacteria.</li>
| |
| − | <ul>A. Buffer PI was spilled on the table, but another bottle was brought down.</ul>
| |
| − | <ul>B. To thoroughly mix it, vortex the microcentrifuge column.</ul>
| |
| − | <li>Add 250 microlitres of Buffer P2. Mix by inverting it 4-6 times.</li>
| |
| − | <ul>A. Don’t let this sit for more than 5 minutes.</ul>
| |
| − | <li>Add 350 microlitres of Buffer N3 and mix by inverting 4-6 times.</li>
| |
| − | <ul>A. A whitish precipitate should have formed.</ul>
| |
| − | <li>Microcentrifuge for 10 minutes at >10 000 rpm.</li>
| |
| − | <ul>A. Supernatant is what we want. The solid is the garbage.</ul>
| |
| − | <ul>B. Once taken out, supernatant will be pipetted out into a separate tube (not a spin column). This allows unison of the supernatant and consistency</ul>
| |
| − | <ul>C. *Note* Eppendorf tubes are microcentrifuge tubes</ul>
| |
| − | <li>Pipette out supernatant from new microcentrifuge into labeled spin columns</li>
| |
| − | <ul>A. Centrifuge it for 30-60 seconds</ul>
| |
| − | <ul>B. Let it sit for a bit</ul>
| |
| − | <li>Add 0.75 ml of Buffer PE and centrifuge for 30-60s.</li>
| |
| − | <ul>A. Discard flow-through and centrifuge one more time for 1 minute.</ul>
| |
| − | <ul>B. Residual wash buffer (remove it)</ul>
| |
| − | <li>Add 100 microlitres of Buffer EB after transferring spin column over a new microcentrifuge tube.</li>
| |
| − | <ul>A. Stand for one minute</ul>
| |
| − | <ul>B. Spin for one minute</ul>
| |
| − | <ul>C. *Technique* Using finger to balance it and place it just above the filtre- Don’t touch the filter.
| |
| − | </ul>
| |
| − | <li>Now, we will test concentration and purity through the use of a spectrophotometer.</li>
| |
| − | <li>Results:</li>
| |
| − | <table style="width:96%">
| |
| − | <tr>
| |
| − | <th></th>
| |
| − | <th>Vial Number</th>
| |
| − | <th>260/280</th>
| |
| − | <th>Concentration in nanograms/microlitre</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>1</td>
| |
| − | <td>1.77</td>
| |
| − | <td>88.4</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>1</td>
| |
| − | <td>1.80</td>
| |
| − | <td>85.0</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>2</td>
| |
| − | <td>1.81</td>
| |
| − | <td>84.4</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>2</td>
| |
| − | <td>1.80</td>
| |
| − | <td>85.0</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>3</td>
| |
| − | <td>1.79</td>
| |
| − | <td>87.0</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>3</td>
| |
| − | <td>1.81</td>
| |
| − | <td>78.6</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>4</td>
| |
| − | <td>1.79</td>
| |
| − | <td>83.6</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>4</td>
| |
| − | <td>1.79</td>
| |
| − | <td>83.1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>5</td>
| |
| − | <td>1.79</td>
| |
| − | <td>77.7</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>5</td>
| |
| − | <td>1.77</td>
| |
| − | <td>81.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>6</td>
| |
| − | <td>1.81</td>
| |
| − | <td>76.7</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>6</td>
| |
| − | <td>1.79</td>
| |
| − | <td>77.2</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </ol>
| |
| − |
| |
| − | <br><hr></hr></br>
| |
| − |
| |
| − | <h3>July 28th 2016</h3>
| |
| − | <ol>
| |
| − | <p><b>Miniprep (again) today</b></p>
| |
| − | <li>Cld + cultures are ready (not purple this time)</li>
| |
| − | <ul>A. Perform miniprep on them</ul>
| |
| − | <ul>B. We went through the exact procedure as the previous day</ul>
| |
| − | </ol>
| |
| − |
| |
| − | <h3>Miniprep</h3>
| |
| − | <ol>
| |
| − | <li>Observations: Like yesterday, the pellet that formed after the microcentrifugation is reddish-orange</li>
| |
| − | <ul>A. Vial 4 (after vortex with Buffer PI) appears to be purplish in colour rather than pink like the rest.</ul>
| |
| − | <ul>B. Pink: “Strawberry Milkshake”</ul>
| |
| − | <ul>C. Purple: “Glove coloured purple with a slight tint of pink”</ul>
| |
| − | <li>Second centrifugation (with PE) of number 4: the waste was blue and this might be due to the blue dye of the sharpie we used to differentiate vial 4’s content from the rest.</li>
| |
| − | <ul>A. At first, it was believed this was due to the initial purple colour, but upon closer inspection, it was realized some blue streaked marks suggested that enthanol cleaned away the blue sharpie dye.</ul>
| |
| − | <li>Results:</li>
| |
| − | <table style="width:96%">
| |
| − | <tr>
| |
| − | <th></th>
| |
| − | <th>Vial Number</th>
| |
| − | <th>260/280</th>
| |
| − | <th>Concentration in nanograms/microlitre</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>1</td>
| |
| − | <td>1.78</td>
| |
| − | <td>86.7</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>1</td>
| |
| − | <td>1.83</td>
| |
| − | <td>85.4</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>2</td>
| |
| − | <td>1.79</td>
| |
| − | <td>98.5</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>2</td>
| |
| − | <td>1.79</td>
| |
| − | <td>98.6</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>3</td>
| |
| − | <td>1.83</td>
| |
| − | <td>81.9</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>3</td>
| |
| − | <td>N/A</td>
| |
| − | <td>N/A</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 3</td>
| |
| − | <td>3</td>
| |
| − | <td>1.82</td>
| |
| − | <td>82.9</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>4</td>
| |
| − | <td>1.82</td>
| |
| − | <td>97.1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>4</td>
| |
| − | <td>1.80</td>
| |
| − | <td>97.3</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>5</td>
| |
| − | <td>1.82</td>
| |
| − | <td>92.7</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>5</td>
| |
| − | <td>1.82</td>
| |
| − | <td>93.9</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 1</td>
| |
| − | <td>6</td>
| |
| − | <td>1.82</td>
| |
| − | <td>95.4</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Read 2</td>
| |
| − | <td>6</td>
| |
| − | <td>1.82</td>
| |
| − | <td>95.4</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | <ul>A. *N/A is due to pipette contamination. The 260/280 value here was 1.64 and the concentration reading was 93.8 nanograms/microlitre</ul>
| |
| − | <ul>B. Cld+ may have higher concentrations due to histidines present in the Cld+ sequence.</ul>
| |
| − | <li>Next, we created a recipe for mixmaster:</li>
| |
| − | <ul>A. It was calculated through the formula C1V1=C2V2 that the it was required 26.666… or 27 microlitres of cutsmart. 171 microlitres of milliculewater and 2 microlitres of BsaI.</ul>
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| − | <li>Gel Electrophoresis</li>
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| − | <ul>A. To check the plasmid backbone and the Cld +/- insert. </ul>
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| − | <ul>B. Separate (and no gluing)</ul>
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| − | <li>Create master mix by adding 27 microlitres of cutsmart, 171 microlitres of millicule water and 2 microlitres of BsaI. *remember that enzymes are always kept on ice!*</li>
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| − | <li>Transfer fifteen microlitres of master mix into each digestion tube (a total of 13 tubes)</li>
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| − | <ul>A. Each tube is labeled with a digit from 1 to 6 and whether it was Cld - or +. </ul>
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| − | <li>Add 5 microlitres of DNA to each tube.</li>
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| − | <ul>A. *note: cutsmart buffer is used to provide optimal environment for cut enzyme.</ul>
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| − | <ul>B. Cut DNA to compare relative sizes: we will see:</ul>
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| − | <ul>a. Cld +/- (1 Kb) </ul>
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| − | <ul>b. Colour gene (0.7 Kb)</ul>
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| − | <ul>c. Whole plasmid backbone (4 Kb)</ul>
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| − | <b>~Lunch break~</b>
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| − | <li>Gel electrophoresis</li>
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| − | <ul>A. Make gel: 1% agarose in TAE into well</ul>
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| − | <ul>a. *1% is 1g/100ml</ul>
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| − | <ul>B. Add loading dye into tubes</ul>
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| − | <ul>C. Add 5 microlitres of sample into wells</ul>
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| − | <ul>D. Same procedure as practice procedures found online</ul>
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| − | <li>Pcitures of results are taken....</li>
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| − | </ol>
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| − |
| |
| − | <br><hr></hr></br>
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| − |
| |
| − | <h3>August 2nd, 2016</h3>
| |
| − | <ol>
| |
| − | <li>We split the team up into non-science and science groups...</li>
| |
| − | </ol>
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| − |
| |
| − | <br><hr></hr></br>
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| − |
| |
| − | <h3>August 3rd, 2016</h3>
| |
| − | <ol>
| |
| − | <ul>
| |
| − | <li>Science based</li>
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| − | <ul>
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| − | <li>Perchlorate team initiates</li>
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| − | <li>Biobrick team initiates</li>
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| − | <li>Process and quant bio are to design the experiments and must research up on it. (This requires some time)</li>
| |
| − | </ul>
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| − | <li>Non science based</li>
| |
| − | <ul>
| |
| − | <li>Fundraising work was done</li>
| |
| − | </ul>
| |
| − | </ul>
| |
| − | <ul>
| |
| − | <li>Biobrick team: tomorrow, they will perform miniprepe after inoculating bacteria replicated today.</li>
| |
| − | <ul>
| |
| − | <li>Today, chemical transformation was performed (refer to 8.5) to create a lot of plasmids (there is not enough)</li>
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| − | <ul>
| |
| − | <ul>
| |
| − | <li>*When referring, note that ligase was not added</li>
| |
| − | </ul>
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| − | <li>Plasmid that were used were those given from igem (required in order to win gold).</li>
| |
| − | <li>Today, inoculation of grown bacteria will be performed to create cultures for miniprep tomorrow.</li>
| |
| − | </ul>
| |
| − | <li>Chloramphenicol: antibiotic will be added to the inoculated cultures.</li>
| |
| − | <ul>
| |
| − | <li>The given plasmids have a chloramphenicol resistance marker.</li>
| |
| − | </ul>
| |
| − | <li>All in all: the purpose of today for the biobrick team: Create more plasmids because there isn’t enough.</li>
| |
| − | <ul>
| |
| − | <li>Then. create cultures for miniprep.</li>
| |
| − | </ul>
| |
| − | </ul>
| |
| − | </ul>
| |
| − | <ul>
| |
| − | <li><b>Procedure of the Biobrick team:</b></li>
| |
| − | </ul>
| |
| − | <ol>
| |
| − | <li>Take out chemically competent e.coli from freezer and put in ice. 50 microlitres of e.coli into microcentrifuge tube.</li>
| |
| − | <li>DNA are added to cells. Stir with pipette tip, but do not pipette up or down.</li>
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| − | <li>Incubate on ice for 30 minutes.</li>
| |
| − | <li>Heat shock at 42 degrees celsius for 30 seconds.</li>
| |
| − | <li>Incubate for 5 minutes on ice, then add 1 ml of LB.</li>
| |
| − | <li>Incubate for 1 hr at 37 degrees celsius at 250 rpm. (5ml of LB and 5 microlitres of Antibiotic)</li>
| |
| − | <li>2 culture tubes: 5 ml of LB, 15 microlitres of antibiotic, and 100 microlitres of plasmid.</li>
| |
| − | <li>Incubate for whole night.</li>
| |
| − | </ol>
| |
| − |
| |
| − | <br><hr></hr></br>
| |
| − |
| |
| − | <p><strong> </strong></p>
| |
| − | <p><strong> August 4th, 2016</strong></p>
| |
| − | <ul>
| |
| − | <li>Inoculation from yesterday failed</li>
| |
| − | <li>Therefore, we must do it again</li>
| |
| − | <ul>
| |
| − | <li>Two tubes: one of each has: 10 microlitres of plasmid and 5 microlitres of antibiotic</li>
| |
| − | </ul>
| |
| − | </ul>
| |
| − | <ol>
| |
| − | <li>10 microlitres of plasmid into 1 microcentrifuge tube</li>
| |
| − | <li>Incubate on ice for 30 minutes</li>
| |
| − | <li>Heat shock at 42 degrees celsius for 90 seconds</li>
| |
| − | <li>Incubate on ice for 5 minutes</li>
| |
| − | <li>Add 1 ml of LB broth into tube and incubate at 37 degrees celsius for 1 hour.</li>
| |
| − | <li>Add 5 ml of LB broth into 2 culture tubes</li>
| |
| − | <li>Add 5 microlitres of antibiotic into each culture tubes</li>
| |
| − | <li>Transfer 200 microlitres of plasmid and e.coli into each culture tubes (Plasmid is called pSBIC3)
| |
| − | </li>
| |
| − | <li>Shake overnight at 37 degrees celsius. </li>
| |
| − | </ol>
| |
| − |
| |
| − | <br><hr></hr></br>
| |
| − |
| |
| − | </ol>
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| − | </div>
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| − | </html> -->
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