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| - | JUNE: WEEK 2 | + | JUNE: WEEK 3 |
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| - | <p><a name="indice"/>
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| - | </p>
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| - | <a name="June.2C_7th"></a><h2> <span class="mw-headline">June, 7th</span></h2>
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| - |
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| - | <p>These BioBrick were resuspended in 15ul ddH20:
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| - | </p>
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| - | <ul><li><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_E2050'>BBa_E2050</A> (mOrange, 744bp) 2010 Kit Plate 2, well 13N in pSB2K3
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| - | </li><li><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K165018'>BBa_K165018</A> (ADH1 terminator, 253 bp) 2010 Kit Plate 3, well 2M, J63009 (Amp)
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| - | </li><li><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K165037'>BBa_K165037</A> (tef2 promoter, 403bp) 2010 Kit Plate 3, well 22O, pSB1AK3
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| - | </li><li><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_P1004'>BBa_P1004</A> (Chloramphenicol resistence cassette, 769 bp) 2009 Kit plate 1, well 7B, pSB1A1
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| - | </li><li><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J61001'>BBa_J61001</A> (R6K origin, 406bp) 2010 Kit plate 1, well 240, pSB1A2
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| - |
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| - | </li><li><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K081008'>BBa_K081008</A> (RBS-luxI, 664bp) 2010 Kit plate 2, well 10L, pSB1A2
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| - | </li><li><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K125500'>BBa_K125500</A> (GFP fusion brick, 718bp) 2010 Kit plate 3, well 2P, pSB1A2
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| - | </li></ul>
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| - | <p>1,5 ul of each culture was transformed in 100 ul of home-made competent <i>E. coli</i> DH5alpha.
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| - | </p><p>Tranformants were all plated on LB agar plates added with Ampicillin, except for <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_E2050'>BBa_E2050</A> (Kanamycin).
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| - | </p><p>500 ml LB+Amp was prepared and 21 LB agar plates + Amp were prepared (500 ml).
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| - | </p>
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| - |
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| - | <div align="right"><small><a href="#indice" title="">^top</a></small></div>
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| - | <a name="June.2C_8th"></a><h2> <span class="mw-headline">June, 8th</span></h2>
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| - | <p>All plates grown overnight at 37°C showed colonies!
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| - | In particular, <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_E2050'>BBa_E2050</A> (grown on LB+Kan), <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K125500'>BBa_K125500</A> (grown on LB+Amp), <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K081008'>BBa_K081008</A> (grown on LB+Amp) and <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K165018'>BBa_K165018</A> (grown on LB+Amp) showed big colonies, well separated on the plate.
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| - |
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| - | <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_P1004'>BBa_P1004</A> (grown on LB+Amp) and <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J61001'>BBa_J61001</A> (grown on LB+Amp) showed many colonies, with big colonies surrounded by small colonies.
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| - | <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K165037'>BBa_K165037</A> (grown on LB+Amp) showed only 11 big colonies.
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| - | </p><p>A colony was picked for each plate and inoculated in 1ml LB+antibiotic.
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| - | </p><p><br />
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| - | </p>
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| - | <center>
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| - | <table width="100%">
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| - | <tr>
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| - | <td>
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| - | <table border="1">
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| - |
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_E2050'>BBa_E2050</A> </td><td> 1ml LB+Kan
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| - | </td></tr>
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K165037'>BBa_K165037</A> </td><td> 1ml LB+Amp
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| - | </td></tr>
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_P1004'>BBa_P1004</A> </td><td> 1ml LB+Amp
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| - |
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| - | </td></tr>
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K125500'>BBa_K125500</A> </td><td> 1ml LB+Amp
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| - | </td></tr>
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K081008'>BBa_K081008</A> </td><td> 1ml LB+Amp
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| - | </td></tr>
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J61001'>BBa_J61001</A> </td><td> 1ml LB+Amp
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| - |
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| - | </td></tr>
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K165018'>BBa_K165018</A> </td><td> 1ml LB+Amp
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| - | </td></tr></table>
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| - | </td>
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| - | <td>
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| - | <table>
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| - | <tr>
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| - | <td>
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| - | </td>
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| - | </tr>
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| - | <tr>
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| - | <td>
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| - |
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| - | <div class="thumb tleft"><div class="thumbinner" style="width:302px;"><img alt="Sara and Nicolò at work" src="https://static.igem.org/mediawiki/2010/thumb/0/0b/UNIPV_attivita_lab2.jpg/300px-UNIPV_attivita_lab2.jpg" width="300" height="225" border="0" class="thumbimage" /> <div class="thumbcaption"><div class="magnify"></div>Sara and Nicolò at work</div></div></div>
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| - | </td>
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| - | </tr>
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| - | </table>
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| - | </td>
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| - | </tr>
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| - | </table>
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| - | </center>
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| - | <p><br />
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| - | <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K165037'>BBa_K165037</A> in pSB1AK3 plasmid was inocultaed both in 1ml LB+Amp and in 1ml LB+Kan for a phenotipic assay.
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| - | </p><p>Cultures were grown for 8 hours at 37°C 220 rpm.
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| - | </p><p>After this time, cultures were all grown and in saturation phase. <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K165037'>BBa_K165037</A> in LB+Kan was also grown, so that this phenotipic assay was positive.
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| - |
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| - | </p><p>Glycerol stocks were prepared for each culture (250ul saturated culture + 750 ul glycerol 80%) and stored at -80°C.
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| - | </p><p>The left culture was re-filled to 5ml with LB+antibiotic and grown overnight at 37°C 200 rpm to be miniprepped tomorrow.
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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_9th"></a><h2> <span class="mw-headline">June, 9th</span></h2>
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| - | <p>Ligation of:
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| - | </p>
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| - | <ul><li>I0: <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K125500'>BBa_K125500</A> (E-S)+<A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_B0015'>BBa_B0015</A> (E-X)
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| - |
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| - | </li><li>I1: <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_E2050'>BBa_E2050</A> (E-S)+<A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K165018'>BBa_K165018</A> (E-X)
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| - | </li><li>I2: <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_P1004'>BBa_P1004</A> (E-S)+<A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_B0015'>BBa_B0015</A> (E-X)
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| - | </li><li>I3: <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K081008'>BBa_K081008</A> (E-S)+<A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_B0015'>BBa_B0015</A> (E-X)
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| - |
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| - | </li></ul>
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| - | <p><br />
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| - | </p><p><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_B0015'>BBa_B0015</A> was accidentally not inoculated yesterday... :(
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| - | Luckily, we had a DNA stock for this part stored at -20°C, so we used this stock for ligations.
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| - | </p><p>Cultures grown overnight at 37°C 220 rpm were all saturated. BioBricks were extracted with MiniPrep kit.
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| - | Surprisingly, after the first centrifugation, we observed that <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_E2050'>BBa_E2050</A> pellet was orange!! This is curious, because this BioBrick expresses mOrange, but it lacks of promoters. Probably, this spurious transcription is due to the promoter of Kanamycin resistance, that is oriented in the same direction.
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| - | </p>
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| - | <table>
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| - | <tr>
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| - | <td>
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| - | <div class="thumb tleft"><div class="thumbinner" style="width:52px;"><img alt="BBa_E2050 orange pellet" src="https://static.igem.org/mediawiki/2010/thumb/0/0d/UNIPV_attivita_lab1.jpg/50px-UNIPV_attivita_lab1.jpg" width="50" height="139" border="0" class="thumbimage" /> <div class="thumbcaption"><div class="magnify"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></div>BBa_E2050 orange pellet</div></div></div>
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| - |
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| - | </td>
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| - | <td>
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| - | <p>After MiniPrep, purified DNA was quantified with NanoDrop.
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| - | </p>
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| - | <table border="1">
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_E2050'>BBa_E2050</A> </td><td> 139,2 ng/ul
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| - | </td></tr>
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K165037'>BBa_K165037</A> </td><td> 181,5 ng/ul
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| - |
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| - | </td></tr>
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_P1004'>BBa_P1004</A> </td><td> 132,8 ng/ul
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| - | </td></tr>
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K125500'>BBa_K125500</A> </td><td> 124,3 ng/ul
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| - | </td></tr>
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K081008'>BBa_K081008</A> </td><td> 138,3 ng/ul
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| - |
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| - | </td></tr>
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J61001'>BBa_J61001</A> </td><td> 122,2 ng/ul
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| - | </td></tr>
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K165018'>BBa_K165018</A> </td><td> 166,3 ng/ul
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| - | </td></tr></table>
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| - | </td>
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| - | </tr>
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| - |
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| - | </table>
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| - | <p>Digestion of:
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| - | </p>
| |
| - | <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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| - |
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| - | </td></tr>
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K125500'>BBa_K125500</A> </td><td> Insert </td><td> 25 </td><td> 16 </td><td> 4,5 </td><td> 1 EcoRI </td><td> 1 SpeI </td><td> 2,5
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| - |
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| - | </td></tr>
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_B0015'>BBa_B0015</A> </td><td> Vector </td><td> 25 </td><td>6,3 </td><td> 14,2 </td><td> 1 EcoRI </td><td> 1 XbaI </td><td> 2,5
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| - |
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| - | </td></tr>
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_E2050'>BBa_E2050</A> </td><td> Insert </td><td> 25 </td><td> 14 </td><td> 6,5 </td><td> 1 EcoRI </td><td> 1 SpeI </td><td> 2,5
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| - |
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| - | </td></tr>
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K165018'>BBa_K165018</A> </td><td> Vector </td><td> 25 </td><td>6 </td><td> 14,5 </td><td> 1 EcoRI </td><td> 1 XbaI </td><td> 2,5
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| - |
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| - | </td></tr>
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_P1004'>BBa_P1004</A> </td><td> Insert </td><td> 25 </td><td> 15 </td><td> 5,5 </td><td> 1 EcoRI </td><td> 1 SpeI </td><td> 2,5
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| - |
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| - | </td></tr>
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| - | <tr>
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| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K081008'>BBa_K081008</A> </td><td> Insert </td><td> 25 </td><td> 14,5 </td><td> 6 </td><td> 1 EcoRI </td><td> 1 SpeI </td><td> 2,5
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| - |
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| - | </td></tr></table>
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| - | <p>Digestions were incubated at 37°C for 3 hours, gel run and gel-extracted.
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| - | </p>
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| - | <table align="center">
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| - | <tr><td>
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| - | <div class="thumb tleft"><div class="thumbinner" style="width:302px;"><img alt="Gel run/cut of cultures" src="https://static.igem.org/mediawiki/2010/thumb/0/0b/UNIPV_attivita_lab3.jpg/300px-UNIPV_attivita_lab3.jpg" width="300" height="168" border="0" class="thumbimage" /><div class="thumbcaption"><div class="magnify"></div>Gel run/cut of cultures</div></div></div>
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| - | </td></tr></table>
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| - | <p><br />
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| - | Ligation of I0, I1, I2 and I3 was performed at 16°C overnight.
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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_10th"></a><h2> <span class="mw-headline">June, 10th</span></h2>
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| - |
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| - | <p>I0, I1, I2 and I3 were tranformed in home made compotent <i>E. coli</i> DH5-alpha and plated on LB+Amp agar plates.
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| - | </p><p>Plates with transformed <i>E.coli</i> were incubated overnight at 37°C 220 rpm.
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| - | </p><p><br />
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| - | The four plates were named:
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| - | </p>
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| - | <ul><li> iGEM 2010 10/06/2010 DH5-alpha I0 LB+Amp
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| - | </li><li> iGEM 2010 10/06/2010 DH5-alpha I1 LB+Amp
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| - | </li><li> iGEM 2010 10/06/2010 DH5-alpha I2 LB+Amp
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| - |
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| - | </li><li> iGEM 2010 10/06/2010 DH5-alpha I3 LB+Amp
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| - | </li></ul>
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| - | <p>and stored at +4°C.
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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_11th"></a><h2> <span class="mw-headline">June, 11th</span></h2>
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| - | <p>All four plates grown overnight showed colonies!!
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| - | Two colonies for each plate were picked and incubated in 1ml LB+Amp. For I2, a phenotipic test was performed: the same two colonies were inoculated both in LB+Amp and LB+Cm. So, 10 cultures were incubated at 37°C 220 rpm for 8 hours:
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| - | </p>
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| - | <table>
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| - | <tr>
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| - | <td> I0-1 in 1ml LB+Amp; </td><td> I0-2 in 1ml LB+Amp
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| - |
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| - | </td></tr>
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| - | <tr>
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| - | <td> I1-1 in 1ml LB+Amp; </td><td> I1-2 in 1ml LB+Amp
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| - | </td></tr>
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| - | <tr>
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| - | <td> I2-1 in 1ml LB+Amp; </td><td> I2-2 in 1ml LB+Amp
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| - | </td></tr>
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| - | <tr>
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| - | <td> I2-1 in 1ml LB+Cm; </td><td> I2-2 in 1ml LB+Cm
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| - |
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| - | </td></tr>
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| - | <tr>
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| - | <td> I3-1 in 1ml LB+Amp; </td><td> I3-2 in 1ml LB+Amp
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| - | </td></tr></table>
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| - | <p>After over-day growth, we observed that the phenotipic test for I2 was positive for both colonies: I2-1 and I2-2 were grown both in LB+Amp and LB+Cm.
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| - | </p><p>8 glycerol stocks were prepared and stored at -80°C. Next week a screening will be performed to select positive colonies.
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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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| - | <p><br /><br />
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| - | </p>
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| - | <table border="0" width="100%" class="menu">
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| - | <tr>
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| - | <td align="left">Previous week</td>
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| - |
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| - | <td align="right"><a href="/Team:UNIPV-Pavia/Calendar/June/settimana3" title="Team:UNIPV-Pavia/Calendar/June/settimana3"> Next week</a></td>
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| - | </tr>
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| - | </table>
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