From 2013.igem.org
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| - | { | + | {"date" : "2013-07-26","author" : "gabriele","title" : "Re-screening","content" : "<html>I performed again the screening on Caterina's samples from 17/07 but instead of AgeI, which is wrong, I used BamHI-HF and PstI-HF. The screening was negative... :(</html>","tags" : "SAMsynthetase"} |
| - | "date" : "2013-07-17",
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| - | "author" : "gabriele-caterina",
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| - | "title" : "A short day",
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| - | "content" : "<html><h3>Screening with AgeI</h3>First, Caterina performed a screening using AgeI on the \"1:1 A\" sample from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-15-gabriele\">15/07</a>.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion mix|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>11A</th></tr><tr><td>template</td><td>4.34µl</td></tr><tr><td>AgeI-HF</td><td rowspan=\"2\">1µl</td></tr><tr><td>EcoRI-HF</td></tr><tr><td>NEBuffer4</td><td rowspan=\"2\">2µl</td></tr><tr><td>BAS</td></tr><tr><td>Water</td><td>9.66µl</td></tr></table></center></html>}}<html>But then we realized the Gabriele was mistaken, AgeI is able to cut both Plac+RFP and SAMsynthetase so it is not the correct enzyme to perform the screening with...<br><hr><h3>Short digestion</h3>Gabriele then performed a short digestion (aka: a digestion similar to the screening run for 2 hours) with the sample G2_EX-SAMsynth-SP (62.6ng/µl from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-15-gabriele\">15/07</a>) and G4A_linear-pSB1C3 (41.9ng/µl from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-01-Gabriele\">01/07</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-SAM-SP</th><th>linear-pSB1C3</th></tr><tr><td>template</td><td>24µl</td><td>23.87µl</td></tr><tr><td>EcoRI-HF</td><td rowspan=\"2\" colspan=\"2\">1µl</td></tr><tr><td>PstI</td></tr><tr><td>NEBuffer2</td><td rowspan=\"2\" colspan=\"2\">3µl</td></tr><tr><td>BSA</td></tr><tr><td>water</td><td>3µl</td><td>3.13µl</td></tr></table></center></html>}}<html>After the 2 hours of incubation, Gabriele added 1µl of DpnI to the G2 sample and 1µl of SAP to the G4A sample and incubated them at 37°C for 1h.<br><hr><h3>Digestions purification</h3>After that, Gabriele purificated both today's short digestion and the O/N digestion from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-17-gabriele-caterina\">yesterday</a>.<center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Digestion</th><th>Quantity</th></tr><tr><td>EX-SAMsynth-SP</td><td>Short</td><td>39.0ng/µl</td></tr><tr><td>linear-pSB1C3</td><td>Short</td><td>12.1ng/µl</td></tr><tr><td>EX-SAMsynth-SP</td><td>O/N</td><td>13.1ng/µl</td></tr><tr><td>linear-pSB1C3</td><td>O/N</td><td>7.8ng/µl</td></tr></table></center><br><hr><h3>Short digest Ligation</h3>So, Gabriele performed a ligation of the short digested samples.<center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>Ctrl</th><th>1:1</th><th>1:2</th><th>1:3</th></tr><tr><td>buffer</td><td colspan=\"4\">3.5µl</td></tr><tr><td>plasmid</td><td colspan=\"4\">5µl</td></tr><tr><td>insert</td><td>0</td><td>8µl</td><td>16µl</td><td>26µl</td></tr><tr><td>Ligase</td><td colspan=\"4\">1µl</td></tr><tr><td>Water</td><td>25.5µl</td><td>17.5µl</td><td>9.5µl</td><td>0</td></tr></table></center>Then the ligation mixes were incubated at room temperature for 2 hours.<br><hr><h3>O/N digest Ligation</h3>Finally, Gabriele performed a ligation of the overnight digested samples.<center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>Ctrl</th><th>1:1</th></tr><tr><td>Buffer</td><td colspan=\"2\">3.5µl</td></tr><tr><td>Plasmid</td><td colspan=\"2\">15µl</td></tr><tr><td>Insert</td><td>0</td><td>15µl</td></tr><tr><td>Ligase</td><td colspan=\"2\">1µl</td></tr><tr><td>Water</td><td>15.5µl</td><td>0.5µl</td></tr></table></center>Then, the ligation mixes were incubated at room temperature for 2 hourse.</html>",
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| - | "tags" : "SAMsynthetase"
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| - | } | + | |
Latest revision as of 10:46, 3 October 2013
{"date" : "2013-07-26","author" : "gabriele","title" : "Re-screening","content" : "I performed again the screening on Caterina's samples from 17/07 but instead of AgeI, which is wrong, I used BamHI-HF and PstI-HF. The screening was negative... :(","tags" : "SAMsynthetase"}
"
