Team:UNITN-Trento/Notebook/Labposts/08/24

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Latest revision as of 09:09, 3 October 2013

{ "date" : "2013-08-10", "author" : "viola-thomas", "title" : "cloning of s04617+EFE+B0015 and AraCpBAD+EFE+B0015 in psb1c3", "content" : "Starting from the overnight digestion of yesterday of the EFE PCR with XbaI and PstI (as insertion)and the parts s04617 and AraCpBAD with SpeI and PstI (as destination plasmid)today we treated the isert with DpnI and the plasmids with SAP. We purified the digestions and the final quantification were :

Quatification results

part	initial amount digested	quantification after purification
EFE	all PCR	19.5 ng/µl
AraCpBAD	3µg	38.3 ng/µl
s04617	3µg	13µg

Then we ligate following this protocol. With the part s04617 (pFixK-cI-pLambda) we could do only the 1:2 ligation and the control because of the very small amount of destination vector. Then we transformed the Neb5α cells following the classical transformation protocol.", "tags" : "pFixK-cI-pLambda-EFE-AraCpBAD" }

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 {
 	"date" : "2013-08-10",
-	"author" : "viola-michele-emil",
+	"author" : "viola-thomas",
-	"title" : "Bacillus will not win!!!",
+	"title" : "cloning of s04617+EFE+B0015 and AraCpBAD+EFE+B0015 in psb1c3",
-	"content" : "<html>Today Michele joined the Bacillus' team. We redid 20 ml of fresh transformation media following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#subtilis-transformation\">the Groningen's protocol</a>. Then we did 4 inocula and we waited until they reached the OD 1.00. During this time we performed a digestion with ScaI HF enzyme to tranform pXyl+EFE+B0015 in Bacillus. After an hour of digestion of three different quantities of DNA </html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion with ScaI|<html><table><tr><th></th><th>DNA [&micro;l]</th><th>Buffer [&micro;l]</th><th>ScaI [&micro;l]</th><th>Water [&micro;l]</th><th>Final volume [&micro;l]</th></tr><tr><th>500 ng digestion</th><td>1.27 </th><td>5</td><td>0.5</td><td>43.23</td><td>50</td></tr><tr><th>1000 ng digestion</th><td>2.55 </th><td>5</td><td>0.5</td><td>41.45</td><td>50</td></tr><tr><th>2000 ng digestion</th><td>5.1 </th><td>5</td><td>0.5</td><td>38.9</td><td>50</td></tr></table></html> }}<html>we deactivate the digestion enzyme 20 minutes at 80°, the we added SAP for an hour at 37° and also this was deactivated for 20 minutes at 80°.It was also done an electrophoretic run to verify the result of this digestion:</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel Image|<html><center><img src=\"https://static.igem.org/mediawiki/2013/9/90/Tn-2013_Foto_2.JPG\" /></center></html>}}<html> As you can see from the picture there is a unique band at about 6000 bp (Kapa Universal Ladder was used). We think that the band in the ladder at 5000 bp is not present because it remains under the bigger lane at 4000. When one of the 2 ml inocula reached the right OD we splitted it into 5 aliquotes of 400&micro;l tubes and we transformed the three pXyl+EFE+B0015 digestions and two quantities of pSpac+EFE+B0015... tomorrow we will see.  Unfortunately The first column(the Emil's one) shows two bands, it means that there are two restriction site for Sca1.This fact is possible only if the GFP isn't the E0840 but the E0240,probably they were mistaken the last year by the previus Igem team.So Emil restarted with two PCR to amplify the correct GFP and to linearize the Backbone K823024(Pxyl), Emil followed the <a href='https://2013.igem.org/Team:UNITN-Trento/Protocols#OneTaq-Phu-PCR'>Onetaq+Phusion PCR protocol</a>. </html>",
+	"content" : "<html>Starting from the overnight digestion of yesterday of the EFE PCR with XbaI and PstI (as insertion)and the parts s04617 and AraCpBAD with SpeI and PstI (as destination plasmid)today we treated the isert with DpnI and the plasmids with SAP. We purified the digestions and the final quantification were :</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Quatification results|<html> <table><tr> <th>part</th><th>initial amount digested</th><th>quantification after purification</th></tr><tr><td>EFE</td><td>all PCR</td><td>19.5 ng/&micro;l</td></tr><tr><td>AraCpBAD</td><td>3&micro;g</td><td>38.3 ng/&micro;l</td></tr><tr><td>s04617</td><td>3&micro;g</td><td>13&micro;g</td></tr></table></html> }}<html>Then we ligate following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Ligation\">this protocol</a>. With the part s04617 (pFixK-cI-pLambda) we could do only the 1:2 ligation and the control because of the very small amount of destination vector. Then we transformed the Neb5&alpha; cells following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Competent-cells-transformation\"> the classical transformation protocol</a>.</html>",
-	"tags" : "pXyl-EFE-B0015"
+	"tags" : "pFixK-cI-pLambda-EFE-AraCpBAD"
 }