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Team:UNICAMP-Brazil/Notebooks/September 26
From 2009.igem.org
(→New Strategy / finO and finP) |
(→Solving the recircularization problems: Dephosphorylation with CIAP) |
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====New Strategy / finO and finP==== | ====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 =( | + | *<p style=”text-align:justify;”>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 =(</p> |
| - | * 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. | + | *<p style=”text-align:justify;”>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.</p> |
| - | * 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. | + | *<p style=”text-align:justify;”>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.</p> |
''Marcelo'' | ''Marcelo'' | ||
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| + | ====Recovering More Biobricks==== | ||
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| + | *<p style=”text-align:justify;”>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 [https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Electroporation Protocol 3].</p> | ||
| + | *<p style=”text-align:justify;”>The transformed cells were plated in LB-AMP media, and were grown for an O/N period.</p> | ||
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| + | ''Marcelo and Victor'' | ||
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| + | ====Cre-Recombinase and pSB1A3 - New digestion==== | ||
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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. | ||
| + | * Sadly, it didn't. =/ | ||
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| + | ''Víctor'' | ||
==''' YeastGuard '''== | ==''' YeastGuard '''== | ||
| - | ====Dephosphorylation | + | ====Solving the recircularization problems: Dephosphorylation with CIAP==== |
| + | *<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> |
| - | We did two ligation reactions, one using the phosphorylated vector (control) and another using the dephosphorylated (Protocol 11). | + | *<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> |
| - | *We transformed the electrocompetent E. coli ( | + | *<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'' | ||
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{{:Team:UNICAMP-Brazil/inc_rodape}} | {{:Team:UNICAMP-Brazil/inc_rodape}} | ||
Latest revision as of 03:18, 22 October 2009
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