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| Line 5: |
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| | <h2> Protocols </h2> | | <h2> Protocols </h2> |
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| − | <h5> <b> Making DMSO Competent Cells </b> </h5> | + | <ul> |
| | + | <li><a href="https://static.igem.org/mediawiki/2014/6/67/AaltoHelsinki_Lab_Notebook_May-June.pdf">May-June (lab)</a></li> |
| | + | <li><a href="https://static.igem.org/mediawiki/2014/b/b2/AaltoHelsinki_Lab_Notebook_July.pdf">July (lab)</a></li> |
| | + | <li><a href="https://static.igem.org/mediawiki/2014/f/fc/AaltoHelsinki_Lab_Notebook_August.pdf">August (lab)</a></li> |
| | + | <li><a href="https://static.igem.org/mediawiki/2014/e/ee/AaltoHelsinki_Lab_Notebook_September-October.pdf">September-October (lab)</a></li> |
| | + | </ul> |
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| − | <table cellspacing="0" cellpadding="0" border="1" bgcolor="grey" bordercolor="black" width="80%" align="center">
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| − | <caption-top> <font size="+1"><font face"Arial"><b><u>Table 1</u>: SOB : 1L</b></font></font></caption>
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| | | | |
| − | <thead> <!--En-tête du tableau -->
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| − | <tr>
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| − | <td><b>Final concentration</b></td>
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| − | <td><b>Composent</b></td>
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| − | <td><b>Volume & Mass</b></td>
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| − | </tr>
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| − | </thead>
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| | | | |
| − | <tbody> <!--Corps du tableau-->
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| − | <tr>
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| − | <td>2%</td>
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| − | <td>Bactotryptone</td>
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| − | <td>20g</td>
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| − | </tr>
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| | | | |
| − | <tr>
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| − | <td>0.5%</td>
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| − | <td>Yeast Extract</td>
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| − | <td>5g</td>
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| − | </tr>
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| − |
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| − | <tr>
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| − | <td>10mM</td>
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| − | <td>NaCl</td>
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| − | <td>2mL (5M stock)</td>
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| − | </tr>
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| − |
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| − | <tr>
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| − | <td>2.5mM</td>
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| − | <td>KCl</td>
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| − | <td>2.5mL (1M stock)</td>
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| − | </tr>
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| − |
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| − | <tr>
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| − | <td>10mM</td>
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| − | <td>MgCl2</td>
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| − | <td>10mL (1M stock)</td>
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| − | </tr>
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| − |
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| − | <tr>
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| − | <td>10mM</td>
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| − | <td>MgSO4</td>
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| − | <td>10mL (1M stock)</td>
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| − | </tr>
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| − |
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| − | <tr>
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| − | <td colspan="3"><b>pH media o 7 with NaOH, then autoclave</b></td>
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| − | </tr>
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| − | </tbody>
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| − |
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| − | </table>
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| − | <br>
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| − |
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| − | <p align="left">- Transformation Broth (TB) : 1L</p>
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| − | <!--Tableau TB à insérer -->
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| − |
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| − | <p align="left">1) Mix the Pipes, CaCl2, and KCl in 900 ml of millipore water.
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| − | <br> 2) Add NaOH until pH is 6.7 (Don’t worry, dust disappear after pH adjust)
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| − | <br> 3) Add MnCl2 (see above), stir, adjust volume to 1 L
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| − | <br> 4) Filter sterilize (Filters are in a cardboard box below the BET bench)
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| − | <br> 5) Store at 4C. 1L</p>
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| − | <br>
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| − |
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| − | <p align="left"> <b>DAY ONE :</b>
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| − | <br> 1) Grow 12 ml overnight culture of favorite strain of E. coli in 2XTY (preheat medium at 37°C before inoculation)
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| − | <br> 2) Make SOB and TB. </p>
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| − |
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| − | <p align="left"> <b>DAY TWO :</b>
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| − | <br> 1) Keep 5mL of SOB for initial OD.
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| − | <br> 2) Inoculate 1 L SOB with 12 ml overnight culture.
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| − | <br> 3) Grow culture at 18C (this temperature is really important as we see a 10-fold decrease in competency when we
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| − | grow them at room temperature). </p>
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| − |
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| − | <p align="left"> <b>DAY THREE :</b>
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| − | <br> 1) Grow cells until A600 0.5-0.7.
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| − | <br>
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| − | <br> <i>Subsequent steps should be carried out in the cold room on ice:</i>
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| − | <br> 2) Put flask on ice for 10 minutes, then spin cells down at 2500xg (3350 RPM) in JLA 8.1 rotor
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| − | <br> 3) Pour off supernatant
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| − | <br> 4) Resuspend gently first in 25 ml TB, then add remaining 295 ml (Final 320mL)
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| − | <br> 5) Leave on ice for 5 minutes
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| − | <br> 6) Spin down again at 2500xg for 10 minutes
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| − | <br> 7) Resuspend cells in 40 ml TB
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| − | <br> 8) Add 3 ml of DMSO dropwise while gently shaking [final DMSO concentration is 7%].
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| − | <br> 9) Aliquot in 100 μl aliquots (you will need about 450 pre-chilled 0.5 ml tubes).
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| − | <br> 10) Flash freeze in liquid nitrogen.
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| − | <br> 11) Store at -80C (the lower the rack the better). </p>
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| − |
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| − | <!--Savy : Rédaction de la page 2 du protocole en cours ; mise en place des tableaux à faire (je dois plancher sur le code pour les faire aussi jolie que sur le pdf de Jean!-->
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| − |
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| − | <h5> <b> Selective Medium for Transformation </b> </h5>
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| − |
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| − | <!-- Tableau de composition à insérer seulement !-->
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| | | | |
| | </div> | | </div> |