Team:UNITN-Trento/Notebook/Labposts/07/23

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Latest revision as of 10:37, 3 October 2013

{"date" : "2013-07-09","author" : "emil","title" : " Purification of Inocula (05/7) and re-ligation and re-transformation ","content" : "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 purification protocol these are the results of the quantification.

Quantification

Sample	Quantities
BBa_K823024+BBa_E0840(1:1) 2	267 ng/µl
BBa_K823024+BBa_E0840(1:1) 3	248.6 ng/µl

Afterwards I screened 800 ng of the samples following the screening protocol with EcoR1 HF and Pst1, these are the results of the gel:

Gel order

Sample	Well
BBa_K823024+BBa_E0840(1:1) b	1
BBa_K823024+BBa_E0840(1:1)	2
Ladder 1kb Fermentas	3

Gel image

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/µl,026b=37.6ng/µl,GFPa=15.9ng/µl,GFPb015.8ng/µl,I have done it following the ligation protocol (I have only changed the amaunt of plasmid:300 ng).Afterwards I have transformed and plated on ampicillin agar 8 NEB10β (2 ctrl,2 1:1, 2 1:2, 2 1:3).I have also amplified the 024(2 inocula).","tags" : "K832024-K823026-E0840"}

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+{"date" : "2013-07-09","author" : "emil","title" : " Purification of Inocula (05/7) and re-ligation and re-transformation ","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>","tags" : "K832024-K823026-E0840"}
-	"date" : "2013-07-27",
-	"author" : "fabio",
-	"title" : " the blue ligation: the never ending story !",
-	"content" : "<html> I first added sap to the o/n digestion( third), then purified and quantified : yield, 32,3 ng/ul.Then again I ligated 006 to 016 folowing the protocol and plated 10 ul in 200 ul of neb10b.In the afternoon I made 6 inocula from the second ligation plates!Today I decided also to take a crack at a fourth ligation with a new strategy: using 006 as my plasmid and 016 as the insert!! I need to digest the two part with different enzymes E and X for 006 and E and S for 016! This time I digested 2400 ng for only 5 ours, not all the night: yields are, 25 ng/ul for 016 and 11,8ng/ul for oo6. Tomorrow I will continue with the ligation number 4.</html>",
-	"tags" : " YF1_FixJ - FixK2"
-}