Team:UNITN-Trento/Notebook/Labposts/06/39

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Latest revision as of 07:51, 3 October 2013

{ "date" : "2013-06-19", "author" : "thomas-viola", "title" : "Minipreps and screening of EFE (in pSB1C3) and AraCpBAD", "content" : "Starting from the inocula of yesterday, we did minipreps followingthis protocol.

Quantification results

EFE in pSB1C3	ng/ul	AraCpBAD	ng/ul
1:4 n°1	552,1	1	285,0
1:4 n°2	482,0	2	311,5
1:4 n°3	558,9	3	446,8
1:2 n°1	779,6	4	404,8
1:2 n°2	376,1	5	442,6
1:1 n°1	359,6
1:1 n°2	501,6

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.

Gel Image

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!", "tags" : "EFE-AraCpBAD" }

@@ Line 1: / Line 1: @@
 {
-	"date" : "2013-06-20",
+	"date" : "2013-06-19",
 	"author" : "thomas-viola",
-	"title" : "Cloning of AraCpBAD (in pSB1C3) + EFE (in Puc57)",
+	"title" : "Minipreps and screening of EFE (in pSB1C3) and AraCpBAD",
-	"content" : "<html>We started digesting 500 ng of AraCpBAD in pSB1C3 with the SpeI and PstI and 500 ng of EFE in Puc57 with XbaI and PstI following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">digestion for Biobricks protocol</a>.<br/><br/>For the digested vector (AraCpBAD), we incubated the part for one hour at 37&deg;C with the SAP phosphatase before disactivating the enzymes.<br/><br/>We then preceeded by ligating and transforming the two parts in competent NEB10b cells following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">the ligation protocol for Biobricks </a>.<br/>We decided to do that in duplicate and with different ratios plasmid:insert (ctrl, 1:1, 1:2, 1:4) obtaining so 8 reactions.<br/><b>Results:</b> as you can see from the pictures, we there are only few colonies in the control so we hope our construct is correctly cloned. The next step will be the inocula and the screening test.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|AraCpBAD (in pSB1C3) + EFE (in Puc57) Ctrl|<html><center><img id=\"post_img\" src=\"https://static.igem.org/mediawiki/2013/5/5f/Tn-20130620-Puc57_Ctrl.jpg\" width=\"400px\"/></center></html>}}{{:Team:UNITN-Trento/Templates/Styles/Spoiler|AraCpBAD (in pSB1C3) + EFE (in Puc57) 1:1|<html><center><img id=\"post_img\" src=\"https://static.igem.org/mediawiki/2013/f/f5/Tn-20130620-Puc57_1-1.jpg\" width=\"400px\"/></center></html>}}{{:Team:UNITN-Trento/Templates/Styles/Spoiler|AraCpBAD (in pSB1C3) + EFE (in Puc57) 1:2|<html><center><img id=\"post_img\" src=\"https://static.igem.org/mediawiki/2013/5/5e/Tn-20130620-Puc57_1-2.jpg\" width=\"400px\"/></center></html>}}{{:Team:UNITN-Trento/Templates/Styles/Spoiler|AraCpBAD (in pSB1C3) + EFE (in Puc57) 1:4|<html><center><img id=\"post_img\" src=\"https://static.igem.org/mediawiki/2013/a/ab/Tn-20130621-Puc57_1-4.jpg\" width=\"400px\"/></center></html>}}",
+	"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>",
-	"tags" : "AraCpBAD-EFE"
+	"tags" : "EFE-AraCpBAD"
 }