Team:UNITN-Trento/Notebook/Labposts/07/18

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{"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"}
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"date" : "2013-07-09",
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"author" : "emil",
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"title" : " Purification of Inocula (05/7) and re-ligation and re-transformation ",
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"content" : "<html>Unfortunately only one of the inocula of 05/7 has succeded and shows an incorrect red color(RFP the previous insert) so I decided to purify and quantfy only the 2 attempt to amplify 024.I have purified them following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#miniprep\"> purification protocol </a> these are the results of the quantification.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Quantification|<html><center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantities</th></tr><tr><td>BBa_K823024+BBa_E0840(1:1) 2</td><td>267 ng/&micro;l</td></tr><tr><td>BBa_K823024+BBa_E0840(1:1) 3</td><td>248.6 ng/&micro;l</td></tr></table></center></html>}}<html>Afterwards I screened 800 ng of the samples following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\"> screening protocol </a> with EcoR1 HF and Pst1, these are the results of the gel:</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>BBa_K823024+BBa_E0840(1:1) b</td><td>1</td></tr><tr><td>BBa_K823024+BBa_E0840(1:1) </td><td>2</td></tr><tr><td>Ladder 1kb Fermentas</td><td>3</td></tr></table></html>}}{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel image|<html><img src=\"https://static.igem.org/mediawiki/2013/9/94/Tn-20130709-d.jpg\" /></html>}}<html>No one of the sample has succeded there are only the bands of the backbone and ther aren't visible inserts of any kind, so I decided to re-try the ligation with two couples of 026 and GFP(026a=36.3 ng/&micro;l,026b=37.6ng/&micro;l,GFPa=15.9ng/&micro;l,GFPb015.8ng/&micro;l,I have done it following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Ligation\"> ligation protocol </a>(I have only changed the amaunt of plasmid:300 ng).Afterwards I have transformed and plated on ampicillin agar  8 NEB10&beta; (2 ctrl,2 1:1, 2 1:2, 2 1:3).I have also amplified the 024(2 inocula).</html>",
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"tags" : "K832024-K823026-E0840"
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}
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Latest revision as of 10:36, 3 October 2013

{"date" : "2013-07-05","author" : "emil","title" : " Purification of the second run of Inocula of ligation and re-ligation of the digested products ","content" : " I added 1µl of DPN1 to the insert and 1µl of SAP to the backbones.Then I have purified the inocula following the miniprep protocol.Afterwards I quantified them toghether with the results of the purification of the digestion products(performed following the purification protocol ) with the following results:

Quantification
SampleQuantification
1:1 024+0840 E482.6ng/µl
1:1 024+0840 E700ng/µl
1:1 024+0840373.1ng/µl
1:3 024+0840423.5ng/µl
1:3 024+0840327.8ng/µl
1:1 026+0840293.1ng/µl
1:1 026+08401243ng/µl
1:3 026+0840766.4ng/µl
1:3 026+0840369.1ng/µl
GFP15.9ng/µl
K82302636.3ng/µl
K82302643.7ng/µl
Then I digested 800 ng of each sample with EcoR1 HF and Pst1 following the digestion protocol with the following results:
Gel order
SampleWell
Ladder 1kb Fermentas1
BBa_K823024+BBa_E0840 A3
BBa_K823024+BBa_E0840 B5
BBa_K823024+BBa_E0840 C6
BBa_K823024+BBa_E0840 D7
BBa_K823024+BBa_E0840 E8
BBa_K823026+BBa_E0840 F9
BBa_K823026+BBa_E0840 G10
BBa_K823026+BBa_E0840 H11
BBa_K823026+BBa_E0840 I12
Gel image
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 ligation protocol .
N.B. Remember always to spin the ligation buffer before using
After that I plated the product of the ligation as done before(1:1,1:3,ctrl for each sample) on Ampicillin added LB.","tags" : "K832024-K823026-E0840"}

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