User:Daniela A. Garcia S./Notebook/Modeling UNAM-Genomics Mexico/2010/08/05

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Wet Lab: OmpC/F promoter OmpC transformation reporter The OmpC tranformation from 04/08/10 was succesful, both the positive control BBa_I51020 and BBa_R0082 had colonies, with the BBa_I51020 in a bigger proportion. In order to test the tranformation product nine colonies from the BBa_R0082 tranformation were re-plaqued; the colonies 1 and 2 were also inoculated in liquid culture (Am). OmpF RTTH PCR PCR1 FWD XbaI_RBS_GFP REV OmpF(1) Template: pSB3K3 + GFP PCR2 FWD XbaI_RBS_GFP REV Bluepromoter-Preffix Template: pSB3K3 + GFP PCR3 FWD Preffix REV Suffix Template: pSB3K3 + GFP > RFP Amplification PCR4 FWD Suffix REV OmpF (1) Template: pSB1C3 PCR3 FWD Preffix REV Suffix Template: pSB1C3 General PCR mixture: 4 μL Template(dilution 1:4) 3 μL Primer forward (5mM) 3 μL Primere reverse (5mM) 6 μL HPLC 6 μL 3.3X XL Buffer 3 μL Mg solution 5 μL dNTP's (0.4 mM) Enzyme(RTTH) mixture for PCR Reaction: 52.5 μL HPLC 45 μL 3.3X XL Buffer 2.5 μL RTTH 20 μL for each reaction Program [35 PCR cycles] 94ºC 5min 94ºC 45seg 55ºC 45seg 72ºC 3min 72ºC 10min 4ºC 3min