Team:UNITN-Trento/Notebook/Labposts/07/44

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Latest revision as of 10:42, 3 October 2013

{"date" : "2013-07-20","author" : "thomas","title" : "Digestion of AraC-pBAD + EFE and Venus PCR product","content" : "This is the part two of this experiment.Since both Venus (BBa_K537006) and AraC-pBAD + EFE were in pSB1C3 vector, I had to do a PCR on Venus using primers Prefix Fwd and Suffing Rev following this protocol. The PCR product was then confirmed by an electrophoresis analysis (Venus is 729 bp and KAPA universal Ladder was adopted).

Gel Image

The PCR product was then purified and quantified at the NanoDrop.Once I obtained Venus without his backbone, I proceeded digesting all the PRC Venus product with NgoMIV and PstI and 2-3 ug of AraC-pBAD + EFE with AgeI and PstI following the PCR product digestion protocol. The digestion products were finally purified, quantified and stored at -20°C.

Quantification Results

AraC-pBAD + EFE	33.5 ng/ul
Venus PCR product	22.8 ng/ul

","tags" : "EFE-Venus"}

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+{"date" : "2013-07-20","author" : "thomas","title" : "Digestion of AraC-pBAD + EFE and Venus PCR product","content" : "<html>This is the part two of <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-18-thomas-michele\">this experiment</a>.Since both Venus (BBa_K537006) and AraC-pBAD + EFE were in pSB1C3 vector, I had to do a PCR on Venus using primers Prefix Fwd and  Suffing Rev following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#pSB1C3-linearization-PCR\">this protocol</a>. The PCR product was then confirmed by an electrophoresis analysis (Venus is 729 bp and KAPA universal Ladder was adopted). <br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel Image|<html><center><img src=\"https://static.igem.org/mediawiki/2013/f/fe/Tn-2013_VenusPCR_gel.jpg\" style =\"width: 450px\"></center></html>}}<html><br/>The PCR product was then purified and quantified at the NanoDrop.Once I obtained Venus without his backbone, I proceeded digesting all the PRC Venus product with NgoMIV and PstI and 2-3 ug of AraC-pBAD + EFE with AgeI and PstI following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">the PCR product digestion protocol</a>. The digestion products were finally purified, quantified and stored at -20&deg;C.<br/> </html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Quantification Results|<html><center><table class=\"tn-sp-table\"><tr><td>AraC-pBAD + EFE</td><td>33.5 ng/ul</td></tr><tr><td>Venus PCR product</td><td>22.8 ng/ul</td></tr></table></center></html>}}<html></html>","tags" : "EFE-Venus"}
-	"date" : "2013-07-05",
-	"author" : "emil",
-	"title" : " Purification of the second run of Inocula of ligation and re-ligation of the digested products ",
-	"content" : "<html> I added 1&micro;l of DPN1 to the insert and 1&micro;l of SAP to the backbones.Then I have purified the inocula following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#miniprep\"> miniprep protocol</a>.Afterwards I quantified them toghether with the results of the purification of the digestion products(performed following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Promega-PCR-Gel\"> purification protocol </a>) with the following results:</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Quantification|<html><center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantification</th></tr><tr><td>1:1 024+0840 E</td><td>482.6ng/&micro;l</td></tr><tr><td>1:1 024+0840 E</td><td>700ng/&micro;l</td></tr><tr><td>1:1 024+0840</td><td>373.1ng/&micro;l</td></tr><tr><td>1:3 024+0840</td><td>423.5ng/&micro;l</td></tr><tr><td>1:3 024+0840</td><td>327.8ng/&micro;l</td></tr><tr><td>1:1 026+0840</td><td>293.1ng/&micro;l</td></tr><tr><td>1:1 026+0840</td><td>1243ng/&micro;l</td></tr><tr><td>1:3 026+0840</td><td>766.4ng/&micro;l</td></tr><tr><td>1:3 026+0840</td><td>369.1ng/&micro;l</td></tr><tr><td>GFP</td><td>15.9ng/&micro;l</td></tr><tr><td>K823026</td><td>36.3ng/&micro;l</td></tr><tr><td>K823026</td><td>43.7ng/&micro;l</td></tr></table></center></html>}}<html>Then I digested 800 ng of each sample with EcoR1 HF and Pst1 following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">digestion protocol</a> with the following results:</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel order|<html><center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Well</th></tr><tr><td>Ladder 1kb Fermentas</td><td>1</td></tr><tr><td>BBa_K823024+BBa_E0840 A</td><td>3</td></tr><tr><td>BBa_K823024+BBa_E0840 B</td><td>5</td></tr><tr><td>BBa_K823024+BBa_E0840 C</td><td>6</td></tr><tr><td>BBa_K823024+BBa_E0840 D</td><td>7</td></tr><tr><td>BBa_K823024+BBa_E0840 E</td><td>8</td></tr><tr><td>BBa_K823026+BBa_E0840 F</td><td>9</td></tr><tr><td>BBa_K823026+BBa_E0840 G</td><td>10</td></tr><tr><td>BBa_K823026+BBa_E0840 H</td><td>11</td></tr><tr><td>BBa_K823026+BBa_E0840 I</td><td>12</td></tr></table></center></html>}}{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel image|<html><img src=\"https://static.igem.org/mediawiki/2013/4/4b/Tn-20130705-ET-Ligation_screening2.jpg\" width=\"450\" /></html>}}<html>As we can see no one of the wells shows the right insert in the lower band(there is always the previous insert the RFP + Lac promoter ~ 1200 bp).At the same time I did the ligation of K823024+E0840 and K823026+E0840 previously digested following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Ligation\"> ligation protocol </a>.<br>N.B. Remember always to spin the ligation buffer before using<br> After that I plated the product of the ligation as done before(1:1,1:3,ctrl for each sample) on Ampicillin added LB.</html>",
-	"tags" : "K832024-K823026-E0840"
-}