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| - | <a name="June.2C_24th"></a><h2> <span class="mw-headline">June, 24th</span></h2>
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| - | <p>PhaP-1 and PhaP-2 plates showed colonies!!
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| - | A colony from each plate was picked and used to infect 1ml LB+Kan. Liquid cultures of PhaP-1 and PhaP-2 were grown for 6 hours at 37°C 220 rpm and then used to prepare glycerol stocks. Remaining liquid cultures were re-filled with 5 ml LB+Kan an grown ON at 37°C 220 rpm for tomorrow MiniPrep.
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| - | Glycerol stocks of PhaP-1 and PhaP-2 are stored at -80°C in the <i>iGEM 2010 Registry</i> box.
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| - | </p><p>Ligations were heated at 65°C for 5 minutes to inactivate ligase ad then tranformed in E. coli TOP10 home made competent cells. Plates were incubated ON at 37°C (we decided to incubate plates 5 hours longer than usual, because cell growth was already slower for I6).
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| - | </p>
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| - | <div align="right"><small><a href="#indice" title="">^top</a></small></div>
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| - | <a name="June.2C_25th"></a><h2> <span class="mw-headline">June, 25th</span></h2>
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| - | <p>All plates showed colonies after 19-hour growth at 37°C!!
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| - | </p>
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| - | <ul><li>I7: small green colonies with satellite colonies were observed. Negative colonies are present and can be easily distinguished, because they are red.
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| - | </li><li>I8: green colonies were observed, few negative colonies are pink.
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| - | </li><li>I9: big colonies were observed, some of them are surrounded by satellite colonies.
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| - | </li><li>I10: big colonies were observed, some light pink colonies are present, surrounded by satellite colonies.
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| - | </li></ul>
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| - | <p>These results are encouraging, because they are consistent with our expectations: ligations provide a high metabolic burden to the cell, so growth is slower. <i>Positive</i> green colonies are smaller and not surrounded by satellite colonies. <i>Negative</i> colonies are bigger and surrounded by satellite colonies, confirming that they had a faster growth than our ligations!
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| - | </p><p>Two colonies from each plate were picked and incubated in 1ml LB+Amp.
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| - | </p>
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| - | <table>
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| - | <tr><td width="50%">
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| - | <table border="1" align="center">
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| - | <tr align="center"><td><b>culture</b></td></tr>
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| - | <tr><td>I7-1</td></tr>
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| - | <tr><td>I7-2</td></tr>
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| - | <tr><td>I8-1</td></tr>
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| - | <tr><td>I8-2</td></tr>
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| - | <tr><td>I9-1</td></tr>
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| - | <tr><td>I9-2</td></tr>
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| - | <tr><td>I10-1</td></tr>
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| - | <tr><td>I10-2</td></tr>
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| - | </table>
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| - | </td><td>
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| - | <p>Cultures were grown for 6 hours at 37°C 220 rpm, then glycerol stocks were prepared for each culture. Glycerol stocks are stored at -80°C in <i>iGEM 2010 ligations</i> box.
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| - | </p>
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| - | </td></tr>
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| - | </table>
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| - | <p>PhaP-1 and PhaP-2 cultures were grown. MiniPre was performed, with the following NanoDrop quantifications:
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| - | </p>
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| - | <table border="1">
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| - | <tr>
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| - | <td> PhaP-1 </td><td> 9,6 ng/ul
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| - | </td></tr>
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| - | <tr>
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| - | <td> PhaP-2 </td><td> 4,3 ng/ul
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| - | </td></tr></table>
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| - | <p>DNA quantification was poor, so we decided to perform digestion screening and to repeat Miniprep next week for sequencing.
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| - | Digestion of:
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| - | </p>
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| - | <table border="1">
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| - | <tr>
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| - | <td> <i>Culture</i> </td><td> <i>Kind</i> </td><td> <i>Final reaction volume (ul) </i> </td><td> <i>DNA (ul)</i> </td><td> <i>H20 (ul)</i> </td><td> <i>Enzyme 1</i> </td><td> <i>Enzyme 2</i> </td><td> <i>Buffer H</i>
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| - | </td></tr>
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| - | <tr>
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| - | <td> PhaP-1 </td><td> Screening </td><td> 25 </td><td> 20,5 </td><td> 0 </td><td> 1 EcoRI </td><td> 1 PstI </td><td> 2,5
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| - | </td></tr>
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| - | <tr>
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| - | <td> PhaP-2 </td><td> Screening </td><td> 25 </td><td> 20,5 </td><td> 0 </td><td> 1 EcoRI </td><td> 1 PstI </td><td> 2,5
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| - | </td></tr></table>
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| - | <p>Digestion was performed for 2 hours at 37°C. DNA was then gel run for screening. Both cultures were positive! Sequencing will be performed next week!
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| - | </p>
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| - | <div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:202px;"><img alt="Screening of PhaP-1 and PhaP-2" src="https://static.igem.org/mediawiki/2010/thumb/e/ec/Unipv_screening_PhaP_1_2.jpg/200px-Unipv_screening_PhaP_1_2.jpg" width="200" height="335" border="0" class="thumbimage" /> <div class="thumbcaption"><div class="magnify"></div>Screening of PhaP-1 and PhaP-2</div></div></div></div>
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