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

From 2013.igem.org

(Difference between revisions)
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{
{
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"date" : "2013-07-21",
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"date" : "2013-07-31",
"author" : "thomas",
"author" : "thomas",
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"title" : "Ligation of AraC-pBAD + EFE and Venus",
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"title" : "pSpac + GFP cloning!!!",
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"content" : "<html>This is the part three 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>. The digestion products were ligated together following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Ligation\">this protocol</a>. The vector (AraC-pBAD + EFE) was ligated with 1:0 (control), 1:1 and 1:4 of insert (Venus). The ligation product was then transformed into NEB10b competent cells and plated on Cloramphenicol containing Petri dishes.</html>",
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"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>",
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"tags" : "EFE-Venus"
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"tags" : "pSpac-GFP"
}
}

Revision as of 08:46, 3 October 2013

{ "date" : "2013-07-31", "author" : "thomas", "title" : "pSpac + GFP cloning!!!", "content" : "Today I started a new cloning in order to obtain GFP in the vector pSBBs0K-Pspac (BBa_K823026). 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 Phusion PCR protocol. I used BB_fwd and BB_rev primers to obtain an amplification of the insert. This because the GFP backbone (BBa_E0840) had the same antibiotic resisistance of our destination vector BBa_K823026 (Amp). I obtain a PCR product with a concentration of 146 ng/µl, confirmed by electrophoresis analysis.

Gel image
I continued then with the restriction digestions following this protocol. I digested BBa_K823026 using SpeI and PstI, and PCR product with XbaI and PstI.
The two digestion products were then purified and quantified: GFP PCR 60 ng/µl, BBa_K823026 19 ng/µl. The last step was then the ligation that was performed following this protocol. The ligation products were then plated on CM plates. ", "tags" : "pSpac-GFP" }

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