From 2013.igem.org
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| - | { | + | {"date":"2013-07-02","author":"viola","title":"<html>EFE+B0015</html>","content":"<html>today i digested B015 in pSB1C3 with the enzymes E and X and EFE in pUC57 with E and S. I started a short cloning protocol with this ligation quantities: <table> <tr> <th>LIGATION</th> <th>CTRL</th> <th>1:1</th> <th>1:2</th> <th>1:4</th> </tr> <tr> <th>BUFFER T4</th> <td>2 µl</td> <td>2 µl</td> <td>2 µl</td> <td>2 µl</td> </tr> <tr> <th>PLASMID (B0015)</th> <td>2.5 µl</td> <td>2.5 µl</td> <td>2.5 µl</td> <td>2.5 µl</td> </tr> <tr> <th>INSEERT (EFE) </th> <td>0 µl</td> <td>1.28 µl</td> <td>2.56 µl</td> <td>5.12 µl</td> </tr> <tr> <th>LIGASE T4 </th> <td>1 µl</td> <td>1 µl</td> <td>1 µl</td> <td>1 µl</td> </tr> <tr> <th>H2O </th> <td>14.5 µl</td> <td>13.2 µl</td> <td>11.9 µl</td> <td>10.6 µl</td> </tr> </table> </html>","tags":"EFE"} |
| - | "date" : "2013-07-15",
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| - | "author" : "gabriele",
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| - | "title" : "Let's try again",
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| - | "content" : "<html><h3>What happened to the inocula???</h3>First, I wanted to miniprep 3 inocula... but two were RED!!! So sad, the RFP was present... that's because I forgot to treat the linear plasmid with DpnI :(<br/>I miniprepped the only not-red inoculum, the \"1:1 A\" which had a concentration of 230.5ng/µl. Then I stocked the sample at -20°C. The possible presence of Plac+RFP is a problem since it is nearly as long as the SAM synthetase gene. So, for the screening, I need an enzyme that cuts only one (either SAM synthetase OR Plac+RFP) forming two fragment with aΔlength higher than 500bp (otherwise it is impossible to distinguish the two bands). Then enzyme that we will use is AgeI.<br/><hr><h3>Another digestion</h3>So, I purified the SAM synthetase extracted through PCR on <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-11-gabriele\">11/07</a>.<center><table class=\"tn-sp-table\"><tr><th>sample</th><th>Quantity</th></tr><tr><td>G1</td><td>116.7ng/µl</td></tr><tr><td>G2</td><td>62.6ng/µl</td></tr><tr><td>G3</td><td>82ng/µl</td></tr></table></center>Then I added 1µl of DpnI to the linear pSB1C3 sample (<b>G2A</b> from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-01-Gabriele\">01/07</a>) and incubated it at 37°C for 1 hour. Then I prepared an overnight digestion following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">usual protocol</a>.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>EX-SAMsynth-SP</th><th>linear pSB1C3</th></tr><tr><td>Template</td><td>34.27µl</td><td>50µl</td></tr><tr><td>EcoRI-HF</td><td rowspan=\"2\">2.5µl</td><td rowspan=\"2\">1.5µl</td></tr><tr><td>PstI-HF</td></tr><tr><td>NEBuffer 2</td><td rowspan=\"2\">10µl</td><td rowspan=\"2\">5µl</td></tr><tr><td>BSA</td></tr><tr><td>Water</td><td>40.73µl</td><td>0</td></tr></table></center></html>}}<html></html>",
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| - | "tags" : "SAMsynthetase"
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| - | } | + | |
Latest revision as of 10:33, 3 October 2013
{"date":"2013-07-02","author":"viola","title":"EFE+B0015","content":"today i digested B015 in pSB1C3 with the enzymes E and X and EFE in pUC57 with E and S. I started a short cloning protocol with this ligation quantities:
| LIGATION | CTRL | 1:1 | 1:2 | 1:4 |
| BUFFER T4 | 2 µl | 2 µl | 2 µl | 2 µl |
| PLASMID (B0015) | 2.5 µl | 2.5 µl | 2.5 µl | 2.5 µl |
| INSEERT (EFE) | 0 µl | 1.28 µl | 2.56 µl | 5.12 µl |
| LIGASE T4 | 1 µl | 1 µl | 1 µl | 1 µl |
| H2O | 14.5 µl | 13.2 µl | 11.9 µl | 10.6 µl |
","tags":"EFE"}
"
