Team:UNITN-Trento/Notebook/Labposts/07/62

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Revision as of 08:45, 3 October 2013

{ "date" : "2013-07-30", "author" : "thomas", "title" : "Bacillus transformation!!!", "content" : "In order to test the transformation protocol for B. subtilis that Groeningen team kindly sent to us I prepared a competent cell Media following this protocol. I proceeded then perfonming the transformation using the empty vector BBa_K823026. I finally plated the tranformed cells into LB agar plates with 10 ug/ml of Kanamycin. The day after took a picture of the plate and performed a Gram staining following this protocol.

Plate image

Bacillus microscope image

As you can see from the pictures, seems that Bacillus was successful transformed!!!!", "tags" : "b. subtilis-pspac" }

@@ Line 1: / Line 1: @@
 {
-	"date" : "2013-07-17",
+	"date" : "2013-07-30",
-	"author" : "gabriele-caterina",
+	"author" : "thomas",
-	"title" : "A short day",
+	"title" : "Bacillus transformation!!!",
-	"content" : "<html><h3>Screening with AgeI</h3>First, Caterina performed a screening using AgeI on the \"1:1 A\" sample from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-15-gabriele\">15/07</a>.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion mix|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>11A</th></tr><tr><td>template</td><td>4.34&micro;l</td></tr><tr><td>AgeI-HF</td><td rowspan=\"2\">1&micro;l</td></tr><tr><td>EcoRI-HF</td></tr><tr><td>NEBuffer4</td><td rowspan=\"2\">2&micro;l</td></tr><tr><td>BAS</td></tr><tr><td>Water</td><td>9.66&micro;l</td></tr></table></center></html>}}<html>But then we realized the Gabriele was mistaken, AgeI is able to cut both Plac+RFP and SAMsynthetase so it is not the correct enzyme to perform the screening with...<br><hr><h3>Short digestion</h3>Gabriele then performed a short digestion (aka: a digestion similar to the screening run for 2 hours) with the sample G2_EX-SAMsynth-SP (62.6ng/&micro;l from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-15-gabriele\">15/07</a>) and G4A_linear-pSB1C3 (41.9ng/&micro;l from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-01-Gabriele\">01/07</a>).</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>EX-SAM-SP</th><th>linear-pSB1C3</th></tr><tr><td>template</td><td>24&micro;l</td><td>23.87&micro;l</td></tr><tr><td>EcoRI-HF</td><td rowspan=\"2\" colspan=\"2\">1&micro;l</td></tr><tr><td>PstI</td></tr><tr><td>NEBuffer2</td><td rowspan=\"2\" colspan=\"2\">3&micro;l</td></tr><tr><td>BSA</td></tr><tr><td>water</td><td>3&micro;l</td><td>3.13&micro;l</td></tr></table></center></html>}}<html>After the 2 hours of incubation, Gabriele added 1&micro;l of DpnI to the G2 sample and 1&micro;l of SAP to the G4A sample and incubated them at 37&deg;C for 1h.<br><hr><h3>Digestions purification</h3>After that, Gabriele purificated both today's short digestion and the O/N digestion from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-17-gabriele-caterina\">yesterday</a>.<center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Digestion</th><th>Quantity</th></tr><tr><td>EX-SAMsynth-SP</td><td>Short</td><td>39.0ng/&micro;l</td></tr><tr><td>linear-pSB1C3</td><td>Short</td><td>12.1ng/&micro;l</td></tr><tr><td>EX-SAMsynth-SP</td><td>O/N</td><td>13.1ng/&micro;l</td></tr><tr><td>linear-pSB1C3</td><td>O/N</td><td>7.8ng/&micro;l</td></tr></table></center><br><hr><h3>Short digest Ligation</h3>So, Gabriele performed a ligation of the short digested samples.<center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>Ctrl</th><th>1:1</th><th>1:2</th><th>1:3</th></tr><tr><td>buffer</td><td colspan=\"4\">3.5&micro;l</td></tr><tr><td>plasmid</td><td colspan=\"4\">5&micro;l</td></tr><tr><td>insert</td><td>0</td><td>8&micro;l</td><td>16&micro;l</td><td>26&micro;l</td></tr><tr><td>Ligase</td><td colspan=\"4\">1&micro;l</td></tr><tr><td>Water</td><td>25.5&micro;l</td><td>17.5&micro;l</td><td>9.5&micro;l</td><td>0</td></tr></table></center>Then the ligation mixes were incubated at room temperature for 2 hours.<br><hr><h3>O/N digest Ligation</h3>Finally, Gabriele performed a ligation of the overnight digested samples.<center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>Ctrl</th><th>1:1</th></tr><tr><td>Buffer</td><td colspan=\"2\">3.5&micro;l</td></tr><tr><td>Plasmid</td><td colspan=\"2\">15&micro;l</td></tr><tr><td>Insert</td><td>0</td><td>15&micro;l</td></tr><tr><td>Ligase</td><td colspan=\"2\">1&micro;l</td></tr><tr><td>Water</td><td>15.5&micro;l</td><td>0.5&micro;l</td></tr></table></center>Then, the ligation mixes were incubated at room temperature for 2 hourse.</html>",
+	"content" : "<html>In order to test the transformation protocol for <i>B. subtilis</i> that Groeningen team kindly sent to us I prepared a competent cell Media following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#subtilis-transformation\">this protocol</a>. I proceeded then perfonming the transformation using the empty vector BBa_K823026. I finally plated the tranformed cells into LB agar plates with 10 ug/ml of Kanamycin. The day after took a picture of the plate and performed a Gram staining following <a href=\"\">this protocol</a>. </html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Plate image|<html><center><img src=\"https://static.igem.org/mediawiki/2013/e/ec/Tn-2013_bacillus_transformation_plate_1.jpg\" style =\"width: 450px\"></center></html>}}{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Bacillus microscope image|<html><center><img src=\"https://static.igem.org/mediawiki/2013/6/65/Tn-2013_gram_staining_bacillus.jpg\" style =\"width: 450px\"></center></html>}}<html> As you can see from the pictures, seems that <i>Bacillus</i> was successful transformed!!!!</html>",
-	"tags" : "SAMsynthetase"
+	"tags" : "b. subtilis-pspac"
 }