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| | <p class="text2"></p> | | <p class="text2"></p> |
| | <p class="text3"></p> | | <p class="text3"></p> |
| − | <h3 id="reader" class="sub6">4.3.1. Plate reader</h3>
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| | <p class="text4"> | | <p class="text4"> |
| − | 1. Prepare 3 over night cultures for each sample Device1〜Device3, Positive control and Negative control in 3 mL LB medium containing chloramphenicol (35 microg / mL) at 37 °C for 17h and shake at 180 rpm.<br> | + | 1. コピペ。<br> |
| − | 2 .Measured the OD590 of each sample and diluted each sample to adjust OD590 within 5% of 0.5.<br> | + | 2. コピペ。<br> |
| − | 3. Set the plate reader to measure GFP.<br> | + | 3. コピペ。<br> |
| − | 4. Take 1 mL of the samples, and centrifuge at 9000x g, 1 min, 4°C.<br> | + | 4. コピペ。<br> |
| − | 5. Remove the supernatants by using P1000 pipette. <br> | + | 5. コピペ。<br> |
| − | 6. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend.<br> | + | 6. コピペ。<br> |
| − | 7. Place 200 μL of each sample into the 96-well plate as described in Table. 3-7-4-1.<br> | + | 7. コピペ。<br> |
| − | 8. Measure the fluorescence intensity with plate reader.<br> | + | 8. コピペ。<br> |
| − | 9. Rotate the 96-well plate 180 degrees horizontally and measure the fluorescence intensity again.<br></p> | + | 9. コピペ。<br> |
| − | <table width="940 px" border="0px">
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| − | <tr>
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| − | <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/f/f7/Tokyo_Tech_Interlab_Table.3-7-4-1.png" width="700px"/>
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| − | </td>
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| − | </tr>
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| − | <tr>
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| − | <td width="940px">
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| − | <h4 align="center" class="fig"><strong>Table. 3-7-4-1.</strong> Position of samples in 96-well plate</h4>
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| − | <td>
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| − | </tr>
| + | |
| − | </table><br>
| + | |
| − | <h3 id="meter" class="sub6">4.3.2. Flow cytometer</h3>
| + | |
| | <p class="text4"> | | <p class="text4"> |
| − | 1. Prepare 3 over night cultures for each sample Device1〜Device3, Positive control and Negative control in 3m LB medium containing chloramphenicol (35 microg / mL) at 37°C for 17h and shake at 180 rpm.<br>
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| − | 2. Start preparing the flow cytometer 1 h before the end of incubation.<br>
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| − | 3. Measure the OD590 and adjust the volume of each sample to centrifuge so that the amount of pellet will be about the same for every sample.<br>
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| − | 4. Centrifuge the samples at 9000x g, 1min, 4°C.<br>
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| − | 5. Remove the supernatants by using P1000 pipette and suspend the samples with 1mL of filtered PBS (phosphate-buffered saline).<br>
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| − | 6. Dispense all of each suspension into a disposable tube through a cell strainer.<br>
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| − | 7. Measure fluorescence intensity with flow cytometer.<br><br>
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| − | <h3 id="How" class="sub5">4.4. How to process the data</h3>
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| − | <h3 id="Pr" class="sub6">4.4.1. Plate reader</h3>
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| − | <p class="text4">
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| − | <strong>-How to draw the calibration curve</strong><br>
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| − | 1. Place 200 μL of various concentrations of sodium fluorescein (500, 375, 250, 125, 50, 25, 10, 5 ng / mL and PBS only) into the 96-well plate in triplicate.<br>
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| − | 2. Measure the fluorescence intensity with the plate reader.<br>
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| − | 3. Rotate the 96-well plate 180 degrees horizontally.<br>
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| − | 4. Measure the fluorescence intensity again.<br>
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| − | 5. Determine the auto-fluorescence of PBS by calculating the arithmetic mean of fluorescence intensity of PBS added in triplicate and use this value as the background fluorescence. <br>
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| − | 6. Subtract background fluorescence from each fluorescence intensity value of each well containing sodium fluorescein.<br>
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| − | 7. Take the arithmetic mean of the three technical replicates of sodium fluorescein of each concentration.<br>
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| − | 8. Draw the calibration curve.<br> </p><br>
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| − | <p class="text4">
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| − | <strong>-How to obtain the absolute unit of fluorescence intensity</strong><br>
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| − | 1. Measure the fluorescence intensity with the plate reader.<br>
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| − | 2. Rotate the 96-well plate 180 degrees horizontally and measure the fluorescence intensity again.<br>
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| − | 3. Calculate the arithmetic mean of these two results.<br>
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| − | 4. Determine the auto-florescence of PBS by calculating the arithmetic mean of fluorescence intensity of PBS added in triplicate and use this value as the background fluorescence.<br>
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| − | 5. Subtract the background fluorescence from each well containing the samples.<br>
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| − | 6. Divide them by the value of OD590 of each sample.<br>
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| − | 7. Calculate the ng / mL fluorescence per OD590 unit by the formula we obtained from drawing the calibration curve.<br>
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| − | <h3 id="Flow cytometer" class="sub6">4.4.2. Flow cytometer</h3>
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| − | <p class="text3">Cells were gated according to the side scatter (SSC) and the forward scatter (FCS) to exclude cell debris and impurities.</p><br><br>
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| − | <h3 id="Individuals" class="sub5">4.5. Individuals responsible for conducting Interlab study</h3>
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| − | <p class="text2">
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| − | Misa Minegishi : Measured the devices and processed the data.<br>
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| − | Yuta Yamazaki : Measured the devices and processed the data.<br>
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| − | Hiraku Tokuma : Created the devices.<br>
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| − | Riku Shinohara: Created the devices.<br>
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| | <h2 id="Reference" class="smalltitle">6.. Reference</h2> | | <h2 id="Reference" class="smalltitle">6.. Reference</h2> |
| | <p class="text">ここにコピペ。<p><br><br><br> | | <p class="text">ここにコピペ。<p><br><br><br> |
1. Introduction
ここにコピペ。
2. Summary of the Experiment
ここにコピペ。
3. Results
ここにコピペ。
4. Discussion
ここにコピペ。
5. Materials and Methods
5.1. Construction
-Strain
All the samples were DH5alpha strain.
-Plasmids
Device 1: J23101 + I13504(pSB1C3)
5.2. Assay Protocol
5.2.1. Protocol1
1. コピペ。
2. コピペ。
3. コピペ。
4. コピペ。
5. コピペ。
6. コピペ。
7. コピペ。
8. コピペ。
9. コピペ。
6.. Reference
ここにコピペ。
