Team:UNITN-Trento/Notebook/Labposts/06/37

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Latest revision as of 07:50, 3 October 2013

{ "date" : "2013-06-18", "author" : "gabriele-emil", "title" : "SAMsynthetase: episode 2
Attack of the Insert", "content" : "This morning we added 1µl of SAP to the pSB1C3 overnight digestion and 1µl of DpN1 to the SAMsynthetase overnight digestion, then both were incubated at 37°C for 1.5 hours.

During these 1.5 hours, we performed the miniprep (protocol) and quantification of the circular pSB1C3 inocula of yesterday.

Circular pSB1C3 Inocula (Gabriele)

Circular pSB1C3 Inocula (Emil)

Circular pSB1C3 quantification results

Sample	Quantity
G1	184.1 ng/µl
G2	187.7 ng/µl
G3	165.7 ng/µl
E1	117.3 ng/µl
E2	105.7 ng/µl
E3	99.0 ng/µl

After the incubation, we purified the digestion mixes using the Wizard® SV Gel and PCR Clean-Up System and then quantified.

Quantification results

Sample	Quantity
linear pSB1C3	30.0 ng/µl
SAMsynthetase G1	40.4 ng/µl
SAMsynthetase E1	32.8 ng/µl

SAMsynthetase E1 was stocked at -20°C.

Then, we performed the ligation of pSB1C3 and SAMsynthetase exploiting the usual protocol.

Ligation mix

	Ctrl	1:1	1:2	1:4
Buffer	2.5µl	3.0µl
Plasmid	10µl
Insert	0	4.14µl	8.28µl	16.56µl
Ligase	2µl
Water	10.5µl	6.36µl	2.22µl	0
Total	25µl

We incubated the ligation mixes for 2 hours at room temperature.

Also, we extracted R0010 promoter (Plac) from the registry (2013 distribution kit, plate 3, well 3H). Unfortunately, we also extracted a part from the same plate and well of the 2012 distribution kit (BBa_K115032) because we mistook the kits.

Finally, we transformed NEB10β competent cells with the usual protocol, we used the following quantities of DNA: 10µl of each ligation product except for the 1:4 ligation, of which we used 15µl, and 1µl of the extracted R0010. Then, we plated on CM plates.", "tags" : "SAMsynthetase" }

@@ Line 1: / Line 1: @@
 {
-	"date" : "2013-06-19",
+	"date" : "2013-06-18",
-	"author" : "thomas-viola",
+	"author" : "gabriele-emil",
-	"title" : "Minipreps and screening of EFE (in pSB1C3) and AraCpBAD",
+	"title" : "SAMsynthetase: episode 2<html><br/></html>''Attack of the Insert''",
-	"content" : "<html>Starting from the inocula of yesterday, we did minipreps following<a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#miniprep\">this protocol</a>.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Quantification results|<html><center><table class=\"tn-sp-table\"><tr><th>EFE in pSB1C3</th><th>ng/ul</th><th>AraCpBAD</th><th>ng/ul</th></tr><tr><td>1:4 n°1</td><td>552,1</td><td>1</td><td>285,0</td></tr><tr><td>1:4 n°2</td><td>482,0</td><td>2</td><td>311,5</td></tr><tr><td>1:4 n°3</td><td>558,9</td><td>3</td><td>446,8</td></tr><tr><td>1:2 n°1</td><td>779,6</td><td>4</td><td>404,8</td></tr><tr><td>1:2 n°2</td><td>376,1</td><td>5</td><td>442,6</td></tr><tr><td>1:1 n°1</td><td>359,6</td></tr><tr><td>1:1 n°2</td><td>501,6</td></tr></table></center></html>}}<html>As you can see we obtained a set of very high concentrations. We proceeded then with the screening test. To do that we digested 900 ng of DNA with EcorI and PstI following this protocol. In the end we prepared the sample for an electrophoresis analysis.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel Image|<html><img id=\"post_img\" src=\"https://static.igem.org/mediawiki/2013/e/ec/Tn-20130619-Thomas-viola_aracpbad_e_efe.jpg\" width=\"500px\"/></html>}}<html>As you can see from the image, al the AraCpBAD samples (on the right of the ladder) were confirmed and 4 out of 7 of EFE were confirmed too..So we have our second Biobrick! EFE in pSB1C3!</html>",
+	"content" : "<html>This morning we added 1&micro;l of SAP to the pSB1C3 overnight digestion and 1&micro;l of DpN1 to the SAMsynthetase overnight digestion, then both were incubated at 37&deg;C for 1.5 hours.<br/><br/>During these 1.5 hours, we performed the miniprep (<a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Promega-PCR-Gel\">protocol</a>) and quantification of the circular pSB1C3 inocula of yesterday.<br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Circular pSB1C3 Inocula (Gabriele)|<html><center><img src=\"https://static.igem.org/mediawiki/2013/5/5e/Tn-20130618-pSB1C3_circular_inocula_gg.JPG\" width=\"450px\" /></center></html>}}<html></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Circular pSB1C3 Inocula (Emil)|<html><center><img src=\"https://static.igem.org/mediawiki/2013/4/4a/Tn-20130618-pSB1C3_circular_inocula_et.JPG\" height=\"450px\" /></center></html>}}<html></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Circular pSB1C3 quantification results|<html><center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantity</th></tr><tr><td>G1</td><td>184.1 ng/&micro;l</td></tr><tr><td>G2</td><td>187.7 ng/&micro;l</td></tr><tr><td>G3</td><td>165.7 ng/&micro;l</td></tr><tr><td>E1</td><td>117.3 ng/&micro;l</td></tr><tr><td>E2</td><td>105.7 ng/&micro;l</td></tr><tr><td>E3</td><td>99.0 ng/&micro;l</td></tr></table></center></html>}}<html><br/>After the incubation, we purified the digestion mixes using the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Promega-PCR-Gel\">Wizard&reg; SV Gel and PCR Clean-Up System</a> and then quantified.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Quantification results|<html><center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantity</th></tr><tr><td>linear pSB1C3</td><td>30.0 ng/&micro;l</td></tr><tr><td>SAMsynthetase G1</td><td>40.4 ng/&micro;l</td></tr><tr><td>SAMsynthetase E1</td><td>32.8 ng/&micro;l</td></tr></table><br/>SAMsynthetase E1 was stocked at -20&deg;C.</center></html>}}<html><br/>Then, we performed the ligation of pSB1C3 and SAMsynthetase exploiting the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Ligation\">usual protocol</a>.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Ligation mix|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border: none;\"></td><th>Ctrl</th><th>1:1</th><th>1:2</th><th>1:4</th></tr><tr><th>Buffer</th><td colspan=\"3\">2.5&micro;l</td><td>3.0&micro;l</td></tr><tr><th>Plasmid</th><td colspan=\"4\">10&micro;l</td></tr><tr><th>Insert</th><td>0</td><td>4.14&micro;l</td><td>8.28&micro;l</td><td>16.56&micro;l</td></tr><tr><th>Ligase</th><td colspan=\"4\">2&micro;l</td></tr><tr><th>Water</th><td>10.5&micro;l</td><td>6.36&micro;l</td><td>2.22&micro;l</td><td>0</td></tr><tr><th>Total</th><td colspan=\"4\">25&micro;l</td></tr></table></center></html>}}<html>We incubated the ligation mixes for 2 hours at room temperature.<br/><br/>Also, we extracted R0010 promoter (Plac) from the registry (2013 distribution kit, plate 3, well 3H). Unfortunately, we also extracted a part from the same plate and well of the 2012 distribution kit (BBa_K115032) because we mistook the kits.<br/><br/>Finally, we transformed NEB10&beta; competent cells with the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Competent-cells-transformation\">usual protocol</a>, we used the following quantities of DNA: 10&micro;l of each ligation product except for the 1:4 ligation, of which we used 15&micro;l, and 1&micro;l of the extracted R0010. Then, we plated on CM plates.</html>",
-	"tags" : "EFE-AraCpBAD"
+	"tags" : "SAMsynthetase"
 }