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Team:UNITN-Trento/Notebook/Labposts/07/31
From 2013.igem.org
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{ | { | ||
| - | "date" : "2013-07- | + | "date" : "2013-07-17", |
| - | "author" : " | + | "author" : "gabriele-caterina", |
| - | "title" : " | + | "title" : "A short day", |
| - | "content" : "<html> | + | "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>", |
| - | "tags" : " | + | "tags" : "SAMsynthetase" |
} | } | ||
Revision as of 08:33, 3 October 2013
{ "date" : "2013-07-17", "author" : "gabriele-caterina", "title" : "A short day", "content" : "
Screening with AgeI
First, Caterina performed a screening using AgeI on the \"1:1 A\" sample from 15/07.Digestion mix
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...| 11A | |
|---|---|
| template | 4.34µl |
| AgeI-HF | 1µl |
| EcoRI-HF | |
| NEBuffer4 | 2µl |
| BAS | |
| Water | 9.66µl |
Short digestion
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 15/07) and G4A_linear-pSB1C3 (41.9ng/µl from 01/07).Digestion mixes
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.| EX-SAM-SP | linear-pSB1C3 | |
|---|---|---|
| template | 24µl | 23.87µl |
| EcoRI-HF | 1µl | |
| PstI | ||
| NEBuffer2 | 3µl | |
| BSA | ||
| water | 3µl | 3.13µl |
Digestions purification
After that, Gabriele purificated both today's short digestion and the O/N digestion from yesterday.| Sample | Digestion | Quantity |
|---|---|---|
| EX-SAMsynth-SP | Short | 39.0ng/µl |
| linear-pSB1C3 | Short | 12.1ng/µl |
| EX-SAMsynth-SP | O/N | 13.1ng/µl |
| linear-pSB1C3 | O/N | 7.8ng/µl |
Short digest Ligation
So, Gabriele performed a ligation of the short digested samples.| Ctrl | 1:1 | 1:2 | 1:3 | |
|---|---|---|---|---|
| buffer | 3.5µl | |||
| plasmid | 5µl | |||
| insert | 0 | 8µl | 16µl | 26µl |
| Ligase | 1µl | |||
| Water | 25.5µl | 17.5µl | 9.5µl | 0 |
O/N digest Ligation
Finally, Gabriele performed a ligation of the overnight digested samples.| Ctrl | 1:1 | |
|---|---|---|
| Buffer | 3.5µl | |
| Plasmid | 15µl | |
| Insert | 0 | 15µl |
| Ligase | 1µl | |
| Water | 15.5µl | 0.5µl |
