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| <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Demonstrate">Demonstrate</a></li> | | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Demonstrate">Demonstrate</a></li> |
| <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Applied_Design">Applied design</a></li> | | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Applied_Design">Applied design</a></li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/InterLab">InterLab</a></li> |
| + | |
| <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Notebook">Notebook</a></li> | | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Notebook">Notebook</a></li> |
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| <a href="#">Parts</a> | | <a href="#">Parts</a> |
| <ul> | | <ul> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Parts">Parts</a></li> |
| <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Basic_Part">Basic parts</a></li> | | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Basic_Part">Basic parts</a></li> |
| <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Composite_Part">Composite parts</a></li> | | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Composite_Part">Composite parts</a></li> |
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| </li> | | </li> |
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− | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/InterLab">InterLab</a></li> | + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Medals">Medals</a></li> |
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| </ul> | | </ul> |
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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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| <p> | | <p> |
− | microplate reader FLUOstar Omega</br>
| + | <ul class=”a”;padding-left:10px> |
− | emission filter: 520 nm</br>
| + | <li>microplate reader FLUOstar Omega</li> |
− | excitation filter: 485 nm
| + | <li>excitation filter: 485 nm</li> |
| + | <li>emission filter: 520 nm</li></ul> |
| </p> | | </p> |
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| <h3>Material</h3> | | <h3>Material</h3> |
| <p> | | <p> |
− | Fluorescein sodium salt</br> | + | <ul class=”a”;padding-left:10px> |
− | 1xPBS</br> | + | <li>Fluorescein sodium salt</li> |
− | Tissue culture testplate (black with flat bottom) | + | <li>1xPBS</li> |
| + | <li>Tissue culture microplate (black with flat bottom)</li></ul> |
| </p> | | </p> |
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| <ol><li>Prepare fluorescein stock solution</li></ol> | | <ol><li>Prepare fluorescein stock solution</li></ol> |
− | <p> | + | <p><ul class=”a”;padding-left:10px> |
− | 1. Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.</br> | + | <li>1. Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.</li> |
− | 2. Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS. </br> | + | <li>2. Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1x PBS. </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 </br></br> | + | <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> |
| </p> | | </p> |
| <ol><li>Serial dilutions</li></ol> | | <ol><li>Serial dilutions</li></ol> |
− | <p> | + | <p><ul class=”a”;padding-left:10 px> |
− | 1. Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12</br> | + | <li>1. Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12.</li> |
− | 2. Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1</br> | + | <li>2. Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1.</li> |
− | 3. Transfer 100 μl of fluorescein stock solution from A1 into A2. </br> | + | <li>3. Transfer 100 μl of fluorescein stock solution from A1 into A2.</li> |
− | 4. Mix A2 by pipetting up and down 3x and transfer 100 μl into A3. </br> | + | <li>4. Mix A2 by pipetting up and down 3x and transfer 100 μl into A3. </li> |
− | 5. Mix A3 by pipetting up and down 3x and transfer 100 μl into A4. </br> | + | <li>5. Mix A3 by pipetting up and down 3x and transfer 100 μl into A4. </li> |
− | 6. Mix A4 by pipetting up and down 3x and transfer 100 μl into A5. </br> | + | <li>6. Mix A4 by pipetting up and down 3x and transfer 100 μl into A5. </li> |
− | 7. Mix A5 by pipetting up and down 3x and transfer 100 μl into A6. </br> | + | <li>7. Mix A5 by pipetting up and down 3x and transfer 100 μl into A6. </li> |
− | 8. Mix A6 by pipetting up and down 3x and transfer 100 μl into A7. </br> | + | <li>8. Mix A6 by pipetting up and down 3x and transfer 100 μl into A7. </li> |
− | 9. Mix A7 by pipetting up and down 3x and transfer 100 μl into A8. </br> | + | <li>9. Mix A7 by pipetting up and down 3x and transfer 100 μl into A8. </li> |
− | 10. Mix A8 by pipetting up and down 3x and transfer 100 μl into A9. </br> | + | <li>10. Mix A8 by pipetting up and down 3x and transfer 100 μl into A9. </li> |
− | 11. Mix A9 by pipetting up and down 3x and transfer 100 μl into A10.</br> | + | <li>11. Mix A9 by pipetting up and down 3x and transfer 100 μl into A10.</li> |
− | 12. Mix A10 by pipetting up and down 3x and transfer 100 μl into A11. </br> | + | <li>12. Mix A10 by pipetting up and down 3x and transfer 100 μl into A11. </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.)</br>
| + | <li>(Caution: Do not to continue serial dilution into column 12.)</li> |
| + | </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> |
| + | |
| | | |
| <br/><br/> | | <br/><br/> |
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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> |
| | | |
| | | |
| <div id="OD600"> | | <div id="OD600"> |
− | <h2>OD600 Reference point</h2> | + | <h2>OD<sub>600</sub> Reference point</h2> |
| | | |
| </div> | | </div> |
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| <h3>Plate reader</h3> | | <h3>Plate reader</h3> |
| | | |
− | <p> | + | <p><ul class=”a”;padding-left:10px> |
− | Thermo Scientific™ Multiskan™ FC Filter-based Microplate Photometer</br> | + | |
− | Filter: 595 nm</br> | + | <li>Thermo Scientific<sup>TM</sup> Multiskan<sup>TM</sup> FC Filter-based Microplate Photometer</li> |
| + | <li>Filter: 595 nm</li> |
| + | </ul> |
| </p> | | </p> |
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| <div class="aaa"></div> | | <div class="aaa"></div> |
| <h3>Material</h3> | | <h3>Material</h3> |
− | <p> | + | <p><ul class=”a”;padding-left:10px> |
− | 1 ml LUDOX</br> | + | <li>1 ml LUDOX</li> |
− | mQH<sub>2</sub>O</br> | + | <li>mQH<sub>2</sub>O </li> |
− | 96 well cell culture plate (clear with flat-bottom) | + | <li>96 well cell culture plate (clear with flat-bottom)</li> |
| </p> | | </p> |
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| | | |
| <p> | | <p> |
− | 1. Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)</br> | + | <ul class="a"> |
− | 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)</br> | + | <li>1. Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette).</li> |
− | 3. Measure absorbance 600 nm of all samples in all standard measurement modes in instrument</br> | + | <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> |
− | 4. Record the data in excel and Import data into Excel ( OD600 reference point tab ) Sheet_1 provided</br> | + | <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> |
| + | </ul> |
| </p> | | </p> |
| + | |
| | | |
| </div> | | </div> |
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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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| <h3>Material</h3> | | <h3>Material</h3> |
| <p> | | <p> |
− | Competent cells ( Escherichia coli strain DH5α)</br> | + | <ul class=”a”; padding-left:10px> |
− | LB (Luria Bertani) media</br> | + | <li>Competent cells (<I>Escherichia coli strain DH5α</I>)</li> |
− | Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 μg /mL)</br> | + | <li>LB (Luria Bertani) media</li> |
− | 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)</br> | + | <li>Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 μg/mL)</li> |
− | Incubator at 37°C</br> | + | <li>50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block <li>light)</li> |
− | 1.5 ml eppendorf tubes for sample storage</br> | + | <li>Incubator at 37°C</li> |
− | Ice bucket with ice</br> | + | <li>1.5 ml eppendorf tubes for sample storage</li> |
− | Pipettes</br> | + | <li>Ice bucket with ice</li> |
− | 96 well plate(cell culture 96 well plate、tissue culture testplate)</br> | + | <li>Pipettes</li> |
− | Devices (from InterLab Measurement Kit):</br> | + | <li>96 well plate(cell culture 96 well plate、tissue culture testplate)</li> |
− | 1. Negative control(BBa_R0040)</br> | + | <li>Devices (from InterLab Measurement Kit):</li> |
− | 2. Positive control(J23151+B0032+E0040+B0010+B0012)</br> | + | <li> 1. Negative control(BBa_R0040)</li> |
− | 3. Test Device 1: J23101+I13504</br> | + | <li> 2. Positive control(J23151+B0032+E0040+B0010+B0012)</li> |
− | 4. Test Device 2: J23106+I13504</br> | + | <li> 3. Test Device 1: J23101+I13504</li> |
− | 5. Test Device 3: J23117+I13504</br> | + | <li> 4. Test Device 2: J23106+I13504</li> |
− | 6. Test Device 4: J23101+BCD2+E0040+B0015</br> | + | <li> 5. Test Device 3: J23117+I13504</li> |
− | 7. Test Device 5: J23106+BCD2+E0040+B0015</br> | + | <li> 6. Test Device 4: J23101+BCD2+E0040+B0015</li> |
− | 8. Test Device 6: J23117+BCD2+E0040+B0015</br> | + | <li> 7. Test Device 5: J23106+BCD2+E0040+B0015</li> |
| + | <li> 8. Test Device 6: J23117+BCD2+E0040+B0015</li> |
| + | </ul> |
| </p> | | </p> |
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| <p> | | <p> |
− | 1. Day 1 : Resuspended each plasmid in plate 7 and transform into Escherichia coli DH5α.</br> | + | <ul class=”a”;padding-left:10 px> |
− | (Transformation protocol is from iGEM)</br> | + | <li>1. Day 1 : Resuspended each plasmid in plate 7 and transform into <I>Escherichia coli DH5α</I>.</li> |
− | 2. Day 2 : Pick 2 colonies from each of plate and inoculate it on 5-10 mL LB medium +Chloramphenicol.Grow the cells overnight (16-18 hours) at 37°C and 170 rpm.</br> | + | <li> (Transformation protocol is from iGEM)</li> |
− | 3. Day 3 : Set instrument to read OD600 (as OD calibration setting)and measure OD600 of the overnight cultures</br> | + | <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> |
− | 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).</br> | + | <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> |
− | 5. Incubate the cultures at 37°C and 170 rpm.</br> | + | <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> |
− | 6. Take 1000 μL samples of the cultures at 0, 2, 4, and 6 hours of incubation and place samples on ice</br> | + | <li>5. Incubate the cultures at 37°C and 170 rpm.</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</br> | + | <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<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> |
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| <!-- Contact --> | | <!-- Contact --> |
− | <section class="contact"> | + | |
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− | </section>
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| <!-- Copyright --> | | <!-- Copyright --> |
| <div class="copyright"> | | <div class="copyright"> |
− | <ul class="menu"> | + | |
− | <li>© 2017 CCU Taiwan iGEM</li><li>Design: <a href="http://html5up.net">HTML5 UP</a></li>
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| </div> | | </div> |
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| </div> | | </div> |
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| + | <!-- Scripts --> |
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| </body> | | </body> |
| </html> | | </html> |