Team:UNITN-Trento/Notebook/Labposts/07/66

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(Difference between revisions)

Latest revision as of 10:47, 3 October 2013

{"date" : "2013-07-28","author" : "thomas","title" : "EFE + Venus Expression test: FAILED!","content" : "Starting from an inoculum, I diluted the cells 1:100 in fresh LB and I waited until O.D.600 of the sample reached 0.5. I induced then the cells (V=5ml) with 25 ul of Arabinose (5mM) and I incubated them into thermoshaker at 37°C for 4 hours. One sample were not induced and used as negative control.After 4 hours I tried to use the trans-UV too see any differences between induced sample and control. Both culture had the same color (not fluorescence) so the experiment failed. However the sample was sent for sequencing so now I'll wait the result before restart all the cloning steps..","tags" : "EFE-Venus"}

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+{"date" : "2013-07-28","author" : "thomas","title" : "EFE + Venus Expression test: FAILED!","content" : "<html>Starting from an inoculum, I diluted the cells 1:100 in fresh LB and I waited until O.D.600 of the sample reached 0.5. I induced then the cells (V=5ml) with 25 ul of Arabinose (5mM) and I incubated them into thermoshaker at 37&deg;C for 4 hours. One sample were not induced and used as negative control.After 4 hours I tried to use the trans-UV too see any differences between induced sample and control. Both culture had the same color (not fluorescence) so the experiment failed. However the sample was sent for sequencing so now I'll wait the result before restart all the cloning steps..</html>","tags" : "EFE-Venus"}
-	"date" : "2013-07-31",
-	"author" : "thomas",
-	"title" : "pSpac + GFP cloning!!!",
-	"content" : "<html>Today I started a new cloning in order to obtain GFP in the vector pSBBs0K-Pspac (<a href=\"http://parts.igem.org/Part:BBa_K823026\">BBa_K823026</a>). This construct will be useful to Emil in order to characterize Pspac at difference concentration of inducer (IPTG). I started performing a PCR on the GFP following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Phusion-PCR\">Phusion PCR protocol</a>. I used BB_fwd and BB_rev primers to obtain an amplification of the insert. This because the GFP backbone (<a href=\"http://parts.igem.org/partsdb/get_part.cgi?part=BBa_E0840\">BBa_E0840</a>) had the same antibiotic resisistance of our destination vector BBa_K823026 (Amp). I obtain a PCR product with a concentration of 146 ng/&micro;l, confirmed by electrophoresis analysis.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel image|<html><center><img src=\"https://static.igem.org/mediawiki/2013/5/5c/Tn-2013_gel_E0840_PCR.jpg\" width=\"450px\" /></center></html>}}<html>  I continued then with the restriction digestions following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">this protocol</a>. I digested BBa_K823026 using SpeI and PstI, and PCR product with XbaI and PstI. <br/>The two digestion products were then purified and quantified: GFP PCR 60 ng/&micro;l, BBa_K823026 19 ng/&micro;l. The last step was then the ligation that was performed following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Ligation\">this protocol</a>. The ligation products were then plated on CM plates. </html>",
-	"tags" : "pSpac-GFP"
-}