Team:UNITN-Trento/Notebook/Labposts/08/06

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Latest revision as of 09:43, 3 October 2013

{ "date" : "2013-08-02", "author" : "Michele", "title" : "Doing a mess", "content" : "So, I didn't believe Cristina when in July she told us: \"In August we're going to work twice than this\" but I had better trust her. Today I did two TLC on four different samples to detect MeSA presence.

TLC Image

As you can see on the image of one of the two TLC only the control (MeSA pure diluited 1:1 in CH2Cl2) ran. These was the third TLC that failed then we decided to avoid more TLC tests. During the day I checked also the growth of Bacillus (link previous post sporulation). In the crazy afternoon after an hard fight with the order in the Lab I performed a screening on GFP (Bba_E0840) to verify if our stock was correct.

Gel Image

As you can is present the band at 878 bp so the screening gave positive results. During the digestion and the electrophoretic run I also did a transforamtion of Neb10β with EFE in pSB1C3. ", "tags" : "GFP-EFE-TLC" }

@@ Line 1: / Line 1: @@
 {
-	"date" : "2013-08-05",
+	"date" : "2013-08-02",
-	"author" : "thomas",
+	"author" : "Michele",
-	"title" : "EFE PCR",
+	"title" : "Doing a mess",
-	"content" : "<html>Today I performed a PCR on EFE in pUC57 vector in order to linearize it and eliminate the vector. To do this I used primers BBa_Fwd and BBa_Rev. The reaction was assembled as follow exploting both OneTaq and Phusion polymerase.<center><table><tr><th>5x One Taq Buffer</th><td>10&micro;l</td></tr><tr><th>Fwd Primer</th><td>1&micro;l</td></tr><tr><th>Rev Primer</th><td>1&micro;l</td></tr><tr><th>10 mM dNTP's</th><td>1&micro;l</td></tr><tr><th>One Taq</th><td>0.25&micro;l</td></tr><tr><th>Phusion</th><td>0.3&micro;l</td></tr><tr><th>Template DNA</th><td>0.7&micro;l (about 500 ng)</td><tr><th>H2O</th><td>35.75&micro;l</td></tr></tr></table></center>When the reaction finished, I performed an electrophoresis analysis in order to confirm the product. Kapa universal ladder was adopted.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel image|<html><center><img src=\"https://static.igem.org/mediawiki/2013/0/0f/Tn-2013_EFE-PCR_GEL.jpg\" width=\"450px\" /></center></html>}}<html> As you can see, the Gel image is quite confusing. First of all, the marker should be diffused in the sample lane (lane 1) as we can see the same band of the ladder but with a more tenuous coloration. Secondly, the thickest band was at about 1200 bp height even if EFE is 1078 bp long. I think the product has to be confirmed again...!</html>",
+	"content" : "<html>So, I didn't believe Cristina when in July she told us: \"In August we're going to work twice than this\" but I had better trust her. Today I did two TLC on four different samples to detect MeSA presence. </html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|TLC Image|<html><center><img src=\"https://2013.igem.org/File:Tn-2013_TLC_Fail.JPG\" width=\"450px\" /></center></html>}}<html> As you can see on the image of one of the two TLC only the control (MeSA pure diluited 1:1 in CH2Cl2) ran. These was the third TLC that failed then we decided to avoid more TLC tests. During the day I checked also the growth of Bacillus (link previous post sporulation). In the crazy afternoon after an hard fight with the order in the Lab I performed a screening on GFP (Bba_E0840) to verify if our stock was correct.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel Image|<html><center><img src=\"https://2013.igem.org/File:Tn-2013_Gel_screening_GFP_BBa_E0840.jpg\" width=\"450px\" /></center></html>}}<html>As you can is present the band at 878 bp so the screening gave positive results. During the digestion and the electrophoretic run I also did a transforamtion of Neb10&beta; with EFE in pSB1C3. </html>",
-	"tags" : "EFE"
+	"tags" : "GFP-EFE-TLC"
 }