Team:UNICAMP-Brazil/Notebooks/September 26

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====Cre-Recombinase and pSB1A3 - New digestion====
====Cre-Recombinase and pSB1A3 - New digestion====
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* Today we repeated both Cre-Recombinase's and pSB1A3's digestion with XbaI and SpeI. Digestion lasted 3 hours.
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* Today we repeated both Cre-Recombinase's and pSB1A3's digestion with ''Xba''I and ''Spe''I. Digestion lasted 3 hours.
* Then, we ran an 1% agarose gel in order to confirm that digestion actually worked.
* Then, we ran an 1% agarose gel in order to confirm that digestion actually worked.
* Sadly, it didn't. =/
* Sadly, it didn't. =/
Line 31: Line 31:
====Solving the recircularization problems: Dephosphorylation with CIAP====
====Solving the recircularization problems: Dephosphorylation with CIAP====
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The dephosphorylation of the vectors theoretically avoids its recircularization. We tried a diferent protocol from the one used by ColiGuard.
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*<p style=”text-align:justify;”>The dephosphorylation of the vectors theoretically avoids its recircularization. We tried a diferent protocol from the one used by ColiGuard.</p>
*<p style=”text-align:justify;”>We performed 5' dephosphorylation of a biobrick vector (previously digested with ''Xba''I and ''Spe''I) with CIAP ([https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/CIAP_Dephosphorylation Protocol 10]) in order to test the reaction efficiency and compare with SAP protocol ([https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/SAP_Dephosphorylation Protocol 9]).</p>
*<p style=”text-align:justify;”>We performed 5' dephosphorylation of a biobrick vector (previously digested with ''Xba''I and ''Spe''I) with CIAP ([https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/CIAP_Dephosphorylation Protocol 10]) in order to test the reaction efficiency and compare with SAP protocol ([https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/SAP_Dephosphorylation Protocol 9]).</p>
*<p style=”text-align:justify;”>We did two ligation reactions, one using the phosphorylated vector (control) and another using the dephosphorylated ([https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/T4_DNA_Ligase Protocol 11]).</p>
*<p style=”text-align:justify;”>We did two ligation reactions, one using the phosphorylated vector (control) and another using the dephosphorylated ([https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/T4_DNA_Ligase Protocol 11]).</p>
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*<p style=”text-align:justify;”>We transformed the electrocompetent ''E. coli'' ([https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Electroporation Protocol 3]) with the ligations and plated in LB+Amp+Kan media.  </p>
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*<p style=”text-align:justify;”>We transformed the electrocompetent ''E. coli'' ([https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Electroporation Protocol 3]) with the ligations and plated in LB-AMP-KAN media.  </p>
''Raíssa and Taís''
''Raíssa and Taís''

Latest revision as of 03:18, 22 October 2009

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ColiGuard

New Strategy / finO and finP

  • We are truly disappointed that we still couldn't make our finO and finP biobricks. We found a lot of issues that must be solved in order to achieve this objective, and we are totally running out of time =(

  • We decided that we are going to leave finO and finP aside for a while, and we will focus on constructing our modified version of Paris 2007 Team's biobricks.

  • We made a whole schedule for that, and we hope we would complete it in a week tops. The next step would be it's insertion into genomic DNA by homologous recombination.

Marcelo

Recovering More Biobricks

  • We started our "new strategy" (related to modifying Paris 2007 team's biobricks) by recovering some biobricks that had now became useful for our project. We ressuspended biobricks BBa_E0840 (GFP device), BBa_I718017 (lox71), BBa_J61000 (chloramphenicol resistance cassette) and BBa_I718016 (lox66), and then transfomed them into electrocompetent E. coli cells, strain DH10B, according to Protocol 3.

  • The transformed cells were plated in LB-AMP media, and were grown for an O/N period.

Marcelo and Victor

Cre-Recombinase and pSB1A3 - New digestion

  • Today we repeated both Cre-Recombinase's and pSB1A3's digestion with XbaI and SpeI. Digestion lasted 3 hours.
  • Then, we ran an 1% agarose gel in order to confirm that digestion actually worked.
  • Sadly, it didn't. =/

Víctor

YeastGuard

Solving the recircularization problems: Dephosphorylation with CIAP

  • The dephosphorylation of the vectors theoretically avoids its recircularization. We tried a diferent protocol from the one used by ColiGuard.

  • We performed 5' dephosphorylation of a biobrick vector (previously digested with XbaI and SpeI) with CIAP (Protocol 10) in order to test the reaction efficiency and compare with SAP protocol (Protocol 9).

  • We did two ligation reactions, one using the phosphorylated vector (control) and another using the dephosphorylated (Protocol 11).

  • We transformed the electrocompetent E. coli (Protocol 3) with the ligations and plated in LB-AMP-KAN media.

Raíssa and Taís





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