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| | <p><a name="indice"/> | | <p><a name="indice"/> |
| | </p> | | </p> |
| - | <a name="June.2C_21st"></a><h2> <span class="mw-headline">June, 21st</span></h2> | + | <a name="June.2C_28th"></a><h2> <span class="mw-headline">June, 28th</span></h2> |
| - | <p>Planning of the activity of the week. | + | <p> |
| - | Freezer cleaning: for each ligation, we chose the correct clone and stored it in the <i>iGEM 2010 ligations</i> box. All these cloned were gel screened and sequenced and are correct!
| + | <b>First time in wet lab</b> |
| - | The colonies we chose are:
| + | |
| - | | + | |
| | </p> | | </p> |
| - | <table align="center" width="30%" border="1">
| |
| - | <tr align="center"><td><b>colony chosen</b></td>
| |
| - | <td><b>ligation name</b></td></tr>
| |
| - | <tr align="center"><td>I0-2</td><td>I0</td></tr>
| |
| - | <tr align="center"><td>I1-2</td><td>I1</td></tr>
| |
| - | <tr align="center"><td>I2-1</td><td>I2</td></tr>
| |
| - | <tr align="center"><td>I3-1</td><td>I3</td></tr>
| |
| | | | |
| - | <tr align="center"><td>I4-2</td><td>I4</td></tr> | + | <p>11 LB agar plates, 35 LB agar + Amp plates and 15 LB agar + Cm 12.5 were prepared.</p> |
| - | <tr align="center"><td>I5-1</td><td>I5</td></tr> | + | |
| - | <tr align="center"><td>I6-2</td><td>I6</td></tr> | + | <p> |
| - | </table> | + | <ul type="circle"> |
| - | <p>Other colonies resulted positives at the screening but were NOT sequenced and are stored in the <i>iGEM 2010 cemetery</i> box. These clones are: I1-1, I2-2, I4-1 and I6-3. | + | <li><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_C0060'>BBa_C0060</A> was resuspended from iGEM 2011 kit distribution, Plate 1, well 4K in 15 μl of ddH<small><sub>2</sub></small>O |
| | + | <li><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_C0061'>BBa_C0061</A> was resuspended from iGEM 2011 kit distribution, Plate 1, well 4M in 15 μl of ddH<small><sub>2</sub></small>O |
| | + | <li><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J04450'>BBa_J04450</A> was resuspended from iGEM 2011 kit distribution, Plate 1, well 5E in 15 μl of ddH<small><sub>2</sub></small>O |
| | + | <li><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K081022'>BBa_K081022</A> was resuspended from iGEM 2011 kit distribution, Plate 2, well 12N in 15 μl of ddH<small><sub>2</sub></small>O |
| | + | </ul> |
| | </p> | | </p> |
| - | <div align="right"><small><a href="#indice" title="">^top</a></small></div>
| |
| | | | |
| - | <a name="June.2C_22nd"></a><h2> <span class="mw-headline">June, 22nd</span></h2> | + | <p> |
| - | <p>Inoculum of I6, <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23118'>BBa_J23118</A>, <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23110'>BBa_J23110</A>, <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23114'>BBa_J23114</A>, <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23116'>BBa_J23116</A> from glycerol stock in 5ml LB+Amp. Cultures were grown ON at 37°C 220rpm.
| + | For each of them 1 μl was transformed in 100 μl of <i>home-made</i> TOP10 competent cells. |
| | + | Transformants were plated on LB agar plates with the right antibiotic and incubated ON at 37°C. |
| | </p> | | </p> |
| | <div align="right"><small><a href="#indice" title="">^top</a></small></div> | | <div align="right"><small><a href="#indice" title="">^top</a></small></div> |
| - | <a name="June.2C_23rd"></a><h2> <span class="mw-headline">June, 23rd</span></h2>
| |
| | | | |
| - | <p>Cultures incubated ON grew all. Plasmids were extracted with MiniPrep.
| + | <a name="June.2C_29th"></a><h2> <span class="mw-headline">June, 29th</span></h2> |
| - | </p><p><br />
| + | |
| - | </p>
| + | |
| - | <div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:302px;"><a href=" /Image:Unipv_digestion_J231xx_I6. jpg" class="image" title="J231xx and I6 digestion" ><img alt="J231xx and I6 digestion" src="https://static.igem.org/mediawiki/2010/thumb/c/c8/Unipv_digestion_J231xx_I6.jpg/300px-Unipv_digestion_J231xx_I6.jpg" width="300" height="142" border="0" class="thumbimage" /></a> < div class="thumbcaption">< div class=" magnify"><a href="/Image:Unipv_digestion_J231xx_I6.jpg" class="internal" title="Ingrandisci"><img src="/wiki/skins/common/images/magnify- clip.png" width="15" height="11" alt="" /></a></div>J231xx and I6 digestion</div></div></div></div> | + | |
| - | <p>After MiniPrep, purified DNA was quantified with NanoDrop.
| + | |
| - | </p> | + | |
| - | <table border="1" align="center">
| + | |
| - | <tr>
| + | |
| - | <td> I6 </td><td> 193 ng/ul
| + | |
| - | </td></tr>
| + | |
| - | <tr>
| + | |
| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23118'>BBa_J23118</A> </td><td> 107,9 ng/ul
| + | |
| - | | + | |
| - | </td></tr>
| + | |
| - | <tr>
| + | |
| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23110'>BBa_J23110</A> </td><td> 83 ng/ul
| + | |
| - | </td></tr>
| + | |
| - | <tr>
| + | |
| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23114'>BBa_J23114</A> </td><td> 89,8 ng/ul
| + | |
| - | </td></tr>
| + | |
| - | <tr>
| + | |
| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23116'>BBa_J23116</A> </td><td> 82,7 ng/ul
| + | |
| | | | |
| - | </td></tr></table> | + | <p> |
| - | <p><br /> | + | In the morning all plates showed colonies (<A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J04450'>BBa_J04450</A> showed light red colonies); for each we picked one colony and inoculated it in 1 ml of liquid LB with the proper antibiotic (ampicillin for <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_C0060'>BBa_C0060</A>, <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_C0061'>BBa_C0061</A> and <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K081022'>BBa_K081022</A>, kanamycin for <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J04450'>BBa_J04450</A>). Cultures were grown at 37°C, 220 rpm. |
| - | Digestion of:
| + | |
| | </p> | | </p> |
| - | <table border="1" align="center">
| |
| - | <tr>
| |
| - | <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>
| |
| | | | |
| - | </td></tr> | + | <p> |
| - | <tr>
| + | In the afternoon glycerol stocks were prepared (750 μl of cultures and 250 μl of 80% glycerol) and stored at - 80°C except for <A HREF='http://partsregistry.org/wiki/index.php/Part: BBa_C0061'> BBa_C0061</A> which was not sufficiently grown (we let it grow until next morning); then cultures were refilled with 5 ml of liquid LB with the proper antibiotic and incubated ON at 37°C, 220 rpm. |
| - | <td> I6 </td><td> Insert </td><td> 25 </td><td> 9,3 </td><td> 11,2 </td><td> 1 XbaI </td><td> 1 PstI </td><td> 2,5
| + | |
| - | | + | |
| - | </td></tr>
| + | |
| - | <tr>
| + | |
| - | <td> I6-bis </td><td> Insert </td><td> 25 </td><td>9,3 </td><td> 11,2 </td><td> 1 XbaI </td><td> 1 PstI </td><td> 2,5
| + | |
| - | | + | |
| - | </td></tr>
| + | |
| - | <tr>
| + | |
| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part: BBa_J23118'> BBa_J23118</A> </td><td> Vector </td><td> 25 </td><td> 9,3 </td><td> 11,2 </td><td> 1 SpeI </td><td> 1 PstI </td><td> 2,5 | + | |
| - | | + | |
| - | </td></tr>
| + | |
| - | <tr>
| + | |
| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23110'>BBa_J23110</A> </td><td> Vector </td><td> 25 </td><td>12 </td><td> 8,5 </td><td> 1 SpeI </td><td> 1 PstI </td><td> 2,5
| + | |
| - | | + | |
| - | </td></tr>
| + | |
| - | <tr>
| + | |
| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23114'>BBa_J23114</A> </td><td> Vector </td><td> 25 </td><td> 11,2 </td><td> 9,3 </td><td> 1 SpeI </td><td> 1 PstI </td><td> 2,5
| + | |
| - | | + | |
| - | </td></tr>
| + | |
| - | <tr>
| + | |
| - | <td> <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23116'>BBa_J23116</A> </td><td> Vector </td><td> 25 </td><td> 12,1 </td><td> 8,4 </td><td> 1 SpeI </td><td> 1 PstI </td><td> 2,5
| + | |
| - | | + | |
| - | </td></tr></table>
| + | |
| - | <p>Digestions were incubated at 37°C for 3 hours, gel run and gel-extracted.
| + | |
| - | </p><p>Ligations were performed ON at 16°C:
| + | |
| | </p> | | </p> |
| - | <ul><li>I7: <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23118'>BBa_J23118</A> (S-P)+ I6 (X-P) | + | <div align="right"><small><a href="#indice" title="">^top</a></small></div> |
| - | </li><li>I8: <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23110'>BBa_J23110</A> (S-P)+ I6 (X-P) | + | |
| - | </li><li>I9: <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23114'>BBa_J23114</A> (S-P)+ I6 (X-P)
| + | |
| - | </li><li>I10: <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23116'>BBa_J23116</A> (S-P)+ I6 (X-P) | + | |
| | | | |
| - | </li></ul> | + | <a name="June.2C_30th"></a><h2> <span class="mw-headline">June, 30th</span></h2> |
| - | <p>NanoDrop quantifications were not reliable, so every ligation was performed with 3ul of vector, 2ul of insert, 3ul of ddH20, 1ul of T4 ligase and 1ul of T4 ligase buffer. | + | <p> |
| - | </p><p>These four promoters from Anderson Promoters Collection were chose for their <i>strength</i>, measured in Arbitrary Units: | + | All cultures were in saturation growth phase; glycerol stock for <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_C0061'>BBa_C0061</A was prepared and stored at -80°C. |
| | + | </p> |
| | + | <p> |
| | + | Plasmids were extracted with MiniPrep kit; purified DNA was quantified with NanoDrop Spectrophotometer: |
| | </p> | | </p> |
| - | <table border="1" align="center">
| |
| - | <tr>
| |
| - | <td> <b>Part</b> </td><td> <b>RFP (a.u.)</b>
| |
| - | </td></tr>
| |
| - | <tr>
| |
| - | <td><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23118'>BBa_J23118</A></td><td>1429
| |
| | | | |
| - | </td></tr> | + | <center> |
| - | <tr>
| + | |
| - | <td><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23110'>BBa_J23110</A></td><td> 844
| + | |
| - | </td></tr>
| + | |
| - | <tr>
| + | |
| - | <td><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23114'>BBa_J23114</A></td><td> 256
| + | |
| - | </td></tr>
| + | |
| - | <tr>
| + | |
| - | <td><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J23116'>BBa_J23116</A></td><td> 396
| + | |
| - | </td></tr>
| + | |
| - | </table>
| + | |
| | | | |
| - | <p>Soon quantitative tests will be performed to quantify each promoter's strength in terms of RPU.
| |
| - | </p><p>Today we received 2 stabs from iGEM HQ for <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K208001'>BBa_K208001</A> (one for each registry location of this part), the Silver-fusion compatible BioBrick part that codes for phasin. This part is in a pSB3K3 plasmid. Bacteria were streaked on LB+Kan (50 ug/ml) agar plates. Cultures were named PhaP-1 and PhaP-2. Plates were incubated ON at 37°C.
| |
| - | </p>
| |
| - | <div align="right"><small><a href="#indice" title="">^top</a></small></div>
| |
| - | <a name="June.2C_24th"></a><h2> <span class="mw-headline">June, 24th</span></h2>
| |
| - | <p>PhaP-1 and PhaP-2 plates showed colonies!!
| |
| - | 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.
| |
| - | Glycerol stocks of PhaP-1 and PhaP-2 are stored at -80°C in the <i>iGEM 2010 Registry</i> box.
| |
| - | </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).
| |
| - | </p>
| |
| | | | |
| - | <div align="right"><small><a href="#indice" title="">^top</a></small></div> | + | <table class="data"> |
| - | <a name="June.2C_25th"></a><h2> <span class="mw-headline">June, 25th</span></h2> | + | <tr> |
| - | <p>All plates showed colonies after 19-hour growth at 37°C!!
| + | <td class="row"><b>Plasmid</b></td> |
| - | </p> | + | <td class="row"><b>DNA (ng/μl)</b></td> |
| - | <ul><li>I7: small green colonies with satellite colonies were observed. Negative colonies are present and can be easily distinguished, because they are red.
| + | </tr> |
| - | </li><li>I8: green colonies were observed, few negative colonies are pink. | + | |
| - | </li><li>I9: big colonies were observed, some of them are surrounded by satellite colonies. | + | |
| - | </li><li>I10: big colonies were observed, some light pink colonies are present, surrounded by satellite colonies.
| + | |
| - | </li></ul>
| + | |
| - | <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!
| + | |
| | | | |
| - | </p><p>Two colonies from each plate were picked and incubated in 1ml LB+Amp. | + | <tr> |
| - | </p>
| + | <td class="row"><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_C0060'>BBa_C0060</A></td> |
| - | <table>
| + | <td class="row">60.8</td> |
| - | <tr><td width="50%">
| + | </tr> |
| - | <table border="1" align="center"> | + | |
| - | <tr align="center"><td><b>culture</b></td></tr>
| + | |
| - | <tr><td>I7-1</td></tr>
| + | |
| - | <tr><td>I7-2</td></tr>
| + | |
| - | <tr><td>I8-1</td></tr>
| + | |
| - | <tr><td>I8-2</td></tr>
| + | |
| - | <tr><td>I9-1</td></tr>
| + | |
| | | | |
| - | <tr><td>I9-2</td></tr>
| |
| - | <tr><td>I10-1</td></tr>
| |
| - | <tr><td>I10-2</td></tr>
| |
| - | </table>
| |
| - | </td><td>
| |
| - | <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.
| |
| - | </p>
| |
| - | </td></tr>
| |
| - | </table>
| |
| - | <p>PhaP-1 and PhaP-2 cultures were grown. MiniPre was performed, with the following NanoDrop quantifications:
| |
| - | </p>
| |
| | | | |
| - | <table border="1">
| + | <tr> |
| - | <tr> | + | <td class="row"><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_C0061'>BBa_C0061</A></td> |
| - | <td> PhaP-1 </td><td> 9,6 ng/ul | + | <td class="row">75.7</td> |
| - | </td></tr>
| + | </tr> |
| - | <tr>
| + | |
| - | <td> PhaP-2 </td><td> 4,3 ng/ul
| + | |
| - | </td></tr></table> | + | |
| - | <p>DNA quantification was poor, so we decided to perform digestion screening and to repeat Miniprep next week for sequencing.
| + | |
| - | Digestion of:
| + | |
| - | </p> | + | |
| - | <table border="1">
| + | |
| - | <tr>
| + | |
| | | | |
| - | <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>
| |
| | | | |
| - | </td></tr>
| + | <tr> |
| - | <tr> | + | <td class="row"><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_K081022'>BBa_K081022</A></td> |
| - | <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 | + | <td class="row">63.6</td> |
| | + | </tr> |
| | | | |
| - | </td></tr>
| |
| - | <tr>
| |
| - | <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
| |
| | | | |
| - | </td></tr></table> | + | <tr> |
| - | <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! | + | <td class="row"><A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_J04450'>BBa_J04450</A></td> |
| | + | <td class="row">17.5</td> |
| | + | </tr> |
| | + | </table> |
| | + | </center> |
| | + | |
| | + | <p> |
| | + | <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_B0030'>BBa_B0030</A>, <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_B0031'>BBa_B0031</A>, <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_B0032'>BBa_B0032</A>, <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_B0034'>BBa_B0034</A>, <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_B0015'>BBa_B0015</A>, <A HREF='http://partsregistry.org/wiki/index.php/Part:pSB4C5'>pSB4C5</A>, <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_I13501'>BBa_I13501</A>, <A HREF='http://partsregistry.org/wiki/index.php/Part:BBa_I13507'>BBa_I13507</A> were inoculated from freezer glycerol stocks and grown ON at 37°C, 220 rpm for MiniPrep. |
| | </p> | | </p> |
| - | <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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