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| <div id="Fluorescein"> | | <div id="Fluorescein"> |
− | <h2>Fluorescein Fluorescence standard curve</h2> | + | <h2> The standard curve of Fluorescein's fluorescence</h2> |
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| </div> | | </div> |
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| <ul class=”a”;padding-left:10px> | | <ul class=”a”;padding-left:10px> |
| <li>microplate reader FLUOstar Omega</li> | | <li>microplate reader FLUOstar Omega</li> |
− | <li>emission filter: 520 nm</li> | + | <li>excitation filter: 485 nm</li> |
− | <li>excitation filter: 485 nm</li></ul> | + | <li>emission filter: 520 nm</li></ul> |
| </p> | | </p> |
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| <li>Fluorescein sodium salt</li> | | <li>Fluorescein sodium salt</li> |
| <li>1xPBS</li> | | <li>1xPBS</li> |
− | <li>Tissue culture testplate (black with flat bottom)</li></ul> | + | <li>Tissue culture microplate (black with flat bottom)</li></ul> |
| </p> | | </p> |
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| <p><ul class=”a”;padding-left:10px> | | <p><ul class=”a”;padding-left:10px> |
| <li>1. Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.</li> | | <li>1. Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.</li> |
− | <li>2. Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS. </li> | + | <li>2. Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1x PBS. </li> |
− | <li>3. Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50 μM </li> | + | <li>3. Dilute the 2x fluorescein stock solution with 1x PBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50 μM </li> |
| </ul> | | </ul> |
| </p> | | </p> |
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| <li>12. Mix A10 by pipetting up and down 3x and transfer 100 μl into A11. </li> | | <li>12. Mix A10 by pipetting up and down 3x and transfer 100 μl into A11. </li> |
| <li>13. Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste. | | <li>13. Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste. |
− | (Caution: Do not to continue serial dilution into column 12.)</li>
| + | <li>(Caution: Do not to continue serial dilution into column 12.)</li> |
| </ul> | | </ul> |
| </p> | | </p> |
− | <ol><li>Repeat serial dilute for Row B、D、E</strong></li></ol> | + | <ol><li>Repeat serial dilute for Row B、D、E.</strong></li></ol> |
− | <ol><li>Measure fluorescence of all samples in all standard measurement modes in instrument Record the data in your notebook</strong></li></ol> | + | <ol><li>Measure fluorescence of all samples in all standard measurement modes in instrument Record the data in your notebook.</strong></li></ol> |
− | <ol><li>Import data into Excel (fluorescein standard curve tab ) Sheet_1 provided</li></ol> | + | <ol><li>Import data into Excel (fluorescein standard curve tab ) Sheet_1 provided.</li></ol> |
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| <h3>Data result</h3> | | <h3>Data result</h3> |
| <br/> | | <br/> |
− | <img src="https://static.igem.org/mediawiki/2017/8/85/FFs_1.png" style="display:block; margin:auto;"><br/><br/> | + | <img src="https://static.igem.org/mediawiki/2017/c/cc/Ccu_interlab_flu_reference_point.png" style="display:block; margin:auto;"><br/><br/> |
− | <img src="https://static.igem.org/mediawiki/2017/a/a4/FFs_2.png" style="display:block; margin:auto;"><br/><br/> | + | <img src="https://static.igem.org/mediawiki/2017/9/9b/Ccu_interlab2.png" style="display:block; margin:auto;"><br/><br/> |
− | <img src="https://static.igem.org/mediawiki/2017/5/5c/FFs_3.png" style="display:block; margin:auto;"><br/> | + | <img src="https://static.igem.org/mediawiki/2017/8/82/Ccu_interlab3.png" style="display:block; margin:auto;"><br/> |
| </div> | | </div> |
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| <div id="OD600"> | | <div id="OD600"> |
− | <h2>OD600 Reference point</h2> | + | <h2>OD<sub>600</sub> Reference point</h2> |
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| </div> | | </div> |
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| <p><ul class=”a”;padding-left:10px> | | <p><ul class=”a”;padding-left:10px> |
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− | <li>Thermo Scientific™ Multiskan™ FC Filter-based Microplate Photometer</li> | + | <li>Thermo Scientific<sup>TM</sup> Multiskan<sup>TM</sup> FC Filter-based Microplate Photometer</li> |
| <li>Filter: 595 nm</li> | | <li>Filter: 595 nm</li> |
| </ul> | | </ul> |
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| <p> | | <p> |
| <ul class="a"> | | <ul class="a"> |
− | <li>1. Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)</li> | + | <li>1. Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette).</li> |
− | <li>2. Add 100 μl of H<sub>2</sub>O into wells A2, B2, C2, D2 (or 1 mL H<sub>2</sub>O into cuvette)</li> | + | <li>2. Add 100 μl of H<sub>2</sub>O into wells A2, B2, C2, D2 (or 1 mL H<sub>2</sub>O into cuvette).</li> |
− | <li>3. Measure absorbance 600 nm of all samples in all standard measurement modes in instrument</li> | + | <li>3. Measure absorbance 600 nm of all samples in all standard measurement modes in instrument.</li> |
− | <li>4. Record the data in excel and Import data into Excel ( OD600 reference point tab ) Sheet_1 provided</li> | + | <li>4. Record the data in excel and Import data into Excel ( OD600 reference point tab ) Sheet_1 provided.</li> |
| </ul> | | </ul> |
| </p> | | </p> |
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| <h3>Data result</h3> | | <h3>Data result</h3> |
| <br/> | | <br/> |
− | <img src="https://static.igem.org/mediawiki/2017/c/cd/Ee.jpeg" style="display:block; margin:auto;"><br/> | + | <img src="https://static.igem.org/mediawiki/2017/e/e5/Ccu_interlab4.png" style="display:block; margin:auto;"><br/> |
| </div> | | </div> |
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| <p> | | <p> |
| <ul class=”a”; padding-left:10px> | | <ul class=”a”; padding-left:10px> |
− | <li>Competent cells ( Escherichia coli strain DH5α)</li> | + | <li>Competent cells (<I>Escherichia coli strain DH5α</I>)</li> |
| <li>LB (Luria Bertani) media</li> | | <li>LB (Luria Bertani) media</li> |
− | <li>Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 μg /mL)</li> | + | <li>Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 μg/mL)</li> |
| <li>50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block <li>light)</li> | | <li>50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block <li>light)</li> |
| <li>Incubator at 37°C</li> | | <li>Incubator at 37°C</li> |
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| <p> | | <p> |
| <ul class=”a”;padding-left:10 px> | | <ul class=”a”;padding-left:10 px> |
− | <li>1. Day 1 : Resuspended each plasmid in plate 7 and transform into Escherichia coli DH5α.</li> | + | <li>1. Day 1 : Resuspended each plasmid in plate 7 and transform into <I>Escherichia coli DH5α</I>.</li> |
| <li> (Transformation protocol is from iGEM)</li> | | <li> (Transformation protocol is from iGEM)</li> |
| <li>2. Day 2 : Pick 2 colonies from each of plate and inoculate it on 5-10 mL LB <li>medium +Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 170 rpm.</li> | | <li>2. Day 2 : Pick 2 colonies from each of plate and inoculate it on 5-10 mL LB <li>medium +Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 170 rpm.</li> |
− | <li>3. Day 3 : Set instrument to read OD600 (as OD calibration setting)and measure OD600 of the overnight cultures</li> | + | <li>3. Day 3 : Set instrument to read OD<sub>600</sub> (as OD calibration setting)and measure OD<sub>600</sub> of the overnight cultures</li> |
− | <li>4. Dilute the cultures to a target OD 600 of 0.02 in 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (covered with foil to block light).</li> | + | <li>4. Dilute the cultures to a target OD<sub>600</sub> of 0.02 in 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (covered with foil to block light).</li> |
| <li>5. Incubate the cultures at 37°C and 170 rpm.</li> | | <li>5. Incubate the cultures at 37°C and 170 rpm.</li> |
− | <li>6. Take 1000 μL samples of the cultures at 0, 2, 4, and 6 hours of incubation and place samples on ice</li> | + | <li>6. Take 1000 μL samples of the cultures at 0, 2, 4, and 6 hours of incubation and place samples on ice.</li> |
− | <li>7. 4 replicates of 100 uL samples were taken from each culture at 0, 2, 4, and 6 hours of incubation and placed in a 96 well plate for OD and fluorescence measurements using the setup described above</li></ul> | + | <li>7. 4 replicates of 100 uL samples were taken from each culture at 0, 2, 4, and 6 hours of incubation and placed in a 96 well plate for OD<sub>600</sub> and fluorescence measurements using the setup described above.</li></ul> |
| </p> | | </p> |
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| <h3>Data result</h3> | | <h3>Data result</h3> |
| <br/> | | <br/> |
− | <img src="https://static.igem.org/mediawiki/2017/f/f7/IL_cell1.jpg" style="display:block; margin:auto;"><br/><br/> | + | <img src="https://static.igem.org/mediawiki/2017/5/5c/Ccu_interlab7.png" style="display:block; margin:auto;"><br/> |
− | <img src="https://static.igem.org/mediawiki/2017/4/41/IL_cell2.jpg" style="display:block; margin:auto;"><br/> | + | <img src="https://static.igem.org/mediawiki/2017/7/7c/Ccu_interlab8.png" style="display:block; margin:auto;"><br/></br> |
| + | <img src="https://static.igem.org/mediawiki/2017/4/48/Ccu_interlab5.png" style="display:block; margin:auto;"><br/><br/> |
| + | <img src="https://static.igem.org/mediawiki/2017/9/9d/Ccu_interlab6.png" style="display:block; margin:auto;"><br/> |
| </div> | | </div> |
| </section> | | </section> |