Team:UNITN-Trento/Notebook/Labposts/07/57

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Latest revision as of 10:45, 3 October 2013

{"date" : "2013-07-24","author" : "gabriele","title" : "Inocula","content" : "I checked the plates prepared by Caterina yesterday.

plate	#colonies	#inocula
ON Ctrl	0	0
ON 1:1	6	5
ON 1:3	0	0
Short Ctrl	0	0
Short 1:1	1	1
Short 1:3	1	1

As you might notice, an O/N 1:3 ligation was never performed, the problem is that Caterina wrote the wrong labels on the plates, so we can't link the plates back to the ligations... anyway, we will keep the denomination of the plates from now onward.","tags" : "SAMsynthetase"}

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+{"date" : "2013-07-24","author" : "gabriele","title" : "Inocula","content" : "<html>I checked the plates prepared by Caterina <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-23-caterina\">yesterday</a>.<br/><center><table class=\"tn-sp-table\"><tr><th>plate</th><th>#colonies</th><th>#inocula</th></tr><tr><td>ON Ctrl</td><td>0</td><td>0</td></tr><tr><td>ON 1:1</td><td>6</td><td>5</td></tr><tr><td>ON 1:3</td><td>0</td><td>0</td></tr><tr><td>Short Ctrl</td><td>0</td><td>0</td></tr><tr><td>Short 1:1</td><td>1</td><td>1</td></tr><tr><td>Short 1:3</td><td>1</td><td>1</td></tr></table></center><br/>As you might notice, an O/N 1:3 ligation was never performed, the problem is that Caterina wrote the wrong labels on the plates, so we can't link the plates back to the ligations... anyway, we will keep the denomination of the plates from now onward.</html>","tags" : "SAMsynthetase"}
-	"date" : "2013-07-11",
-	"author" : "gabriele",
-	"title" : "Extraction (again) and ligation",
-	"content" : "<html><h3>SAM synthetase extraction</h3><a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-10-gabriele-viola\">Last time</a> I determined the correct protocol to extract SAM synthetase from the <i>E. coli</i> using the new plasmid (at the end, the protocol was <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#SAM-extraction-genome-coli\">the usual one</a>). I performed three PCRs (G1, G2, G3) and run the products on 1% agarose gel.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"4\">Loading scheme</th></tr><tr><td>1kb ladder</td><td>G1</td><td>G2</td><td>G3</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/f/f4/Tn-20130711-GG_SAMsynthetase_extraction_EXSP.jpg\" alt=\"Gel\" title=\"Gel\" width=\"450px\" /></center></html>}}<html><br><hr><h3>Ligation</h3>At first, I added 1&micro;l of SAP to pSB1C3 O/N digestion and 1&micro;l of DpnI to SAMsynthetase O/N digestion, and incubated the samples at 37&deg;C for 1.5h. Then I quantified the digestions.<center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantity</th></tr><tr><td>SAM synthetase</td><td>12.2ng/&micro;l</td></tr><tr><td>pSB1C3</td><th>14.0ng/&micro;l</th></tr></table></center><br/>Then I performed the ligation and left the reaction run for 2h at room temperature.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Ligation Mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>CTRL</th><th>1:1</th><th>1:2</th></tr><tr><td>Buffer</td><td colspan=\"3\">4&micro;l</td></tr><tr><td>Plasmid</td><td colspan=\"3\">14.29&micro;l</td></tr><tr><td>Insert</td><td>0</td><td>9.15&micro;l</td><td>18.3&micro;l</td></tr><tr><td>Ligase</td><td colspan=\"3\">1&micro;l</td></tr><tr><td>Water</td><td>20.71&micro;l</td><td>11.56&micro;l</td><td>2.41&micro;l</td></tr></table></center></html>}}<html>Then I transformed the ligation products in NEB10&beta;</html>",
-	"tags" : "SAMsynthetase"
-}