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<li>③Add 100 μl of dH2O into A2, B2, C2, D2</li> | <li>③Add 100 μl of dH2O into A2, B2, C2, D2</li> | ||
<li>④Measure absorbance 600 nm of all samples in all standard measurement models in instrument</li> | <li>④Measure absorbance 600 nm of all samples in all standard measurement models in instrument</li> | ||
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</ul> | </ul> | ||
</li> | </li> | ||
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<li>③ Dilute the 2x fluorescein stock solution using 1xPBS to make a 1x fluorescein solution (final concentration is 50 μM).</li> | <li>③ Dilute the 2x fluorescein stock solution using 1xPBS to make a 1x fluorescein solution (final concentration is 50 μM).</li> | ||
<li>④ *Illustration of serial dilution samples in 96 well plate or cuvettes: value decreases by 2-fold with each column (50% in column 2, 25% in column 3, 12.5% in column 4, etc.)</li> | <li>④ *Illustration of serial dilution samples in 96 well plate or cuvettes: value decreases by 2-fold with each column (50% in column 2, 25% in column 3, 12.5% in column 4, etc.)</li> | ||
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</ul> | </ul> | ||
</li> | </li> | ||
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<li>① Set your instrument to read Abs600 (as OD calibration setting)</li> | <li>① Set your instrument to read Abs600 (as OD calibration setting)</li> | ||
<li>② Measure Abs600 of the overnight cultures</li> | <li>② Measure Abs600 of the overnight cultures</li> | ||
− | <li>③ | + | <li>③ Dilute the cultures to a target Abs600 of 0.02 (see the volume of preloading culture and media in Excel Dilution Calculation sheets) in 12 ml LB medium + Chloramphenicol in 50 mL falcon tube.</li> |
− | + | <li>④ Remove 500uL of each culture for your zero time point and hold these samples on ice. (You should have 16 sample tubes from the time=0 time point)</li> | |
− | <li> | + | <li>⑤ Incubate the cultures at 37°C and 220 rpm.</li> |
− | <li> | + | <li>⑥ Remove 500uL samples of each culture after 2, 4 and 6 hours of growth. Keep samples on ice while completing the additional time points. You should have 16 sample tubes for each time point.</li> |
− | <li> | + | <li>⑦ Set up your four measurement plates: For colony #1 culture, pipet 100 uL samples into wells A, B, C and D of the row for that device. For colony #2, pipet 100uL samples into wells E, F, G, and H of the row for that device.</li> |
− | <li> | + | <li>⑧⑨ Read your plates, taking care to use the exact same settings used for your fluorescein measurement.</li> |
− | <li> | + | |
Revision as of 18:55, 31 October 2017