Team:UNITN-Trento/Notebook/Labposts/07/23

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Revision as of 08:29, 3 October 2013

{ "date" : "2013-07-11", "author" : "gabriele", "title" : "Extraction (again) and ligation", "content" : "

SAM synthetase extraction

Last time I determined the correct protocol to extract SAM synthetase from the E. coli using the new plasmid (at the end, the protocol was the usual one). I performed three PCRs (G1, G2, G3) and run the products on 1% agarose gel.

Gel

Loading scheme
1kb ladder	G1	G2	G3

$\"Gel\"$

Ligation

At first, I added 1µl of SAP to pSB1C3 O/N digestion and 1µl of DpnI to SAMsynthetase O/N digestion, and incubated the samples at 37°C for 1.5h. Then I quantified the digestions.

Sample	Quantity
SAM synthetase	12.2ng/µl
pSB1C3	14.0ng/µl

Then I performed the ligation and left the reaction run for 2h at room temperature.

Ligation Mixes

	CTRL	1:1	1:2
Buffer	4µl
Plasmid	14.29µl
Insert	0	9.15µl	18.3µl
Ligase	1µl
Water	20.71µl	11.56µl	2.41µl

Then I transformed the ligation products in NEB10β", "tags" : "SAMsynthetase" }

@@ Line 1: / Line 1: @@
 {
-	"date" : "2013-07-27",
+	"date" : "2013-07-11",
-	"author" : "fabio",
+	"author" : "gabriele",
-	"title" : " the blue ligation: the never ending story !",
+	"title" : "Extraction (again) and ligation",
-	"content" : "<html> I first added sap to the o/n digestion( third), then purified and quantified : yield, 32,3 ng/ul.Then again I ligated 006 to 016 folowing the protocol and plated 10 ul in 200 ul of neb10b.In the afternoon I made 6 inocula from the second ligation plates!Today I decided also to take a crack at a fourth ligation with a new strategy: using 006 as my plasmid and 016 as the insert!! I need to digest the two part with different enzymes E and X for 006 and E and S for 016! This time I digested 2400 ng for only 5 ours, not all the night: yields are, 25 ng/ul for 016 and 11,8ng/ul for oo6. Tomorrow I will continue with the ligation number 4.</html>",
+	"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" : " YF1_FixJ - FixK2"
+	"tags" : "SAMsynthetase"
 }