User:Daniela A. Garcia S./Notebook/Modeling UNAM-Genomics Mexico/2010/07/29

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Wet Lab: OmpF/C promoters characterization Electrophoresis Gel OmpF PCR_TaqDNApol In order to probe the PCRs reactions an electroforesis gel was made. This was loadded as follows: Ladder positive control B3K3 B3K3 OmpF positive control B1T3 B1T3 OmpF The reactions for B1T3 were sucessful. The next step is to repeat the PCR reaction with the same protocol but this time using the RTTH enzyme. Oligo Design: GFP suffix FWD >BBa_E0240 FWD 5'- GCT CTA GAG c TCA CAC AGG AAA GTA CTA GAT GCG -3' Length: 24 GC content 45.8% MeltTemp: 56.4ºC This primer was desinged to amplified an expression plasmid [OmpFp + GFP] OmpF PCR PCR1. Primer forward: Suffix Primere reverse: Bluepromoter-Preffix Template: pSB3K3 PCR2. Primer forward: Suffix Primere reverse: OmpFpart1-Preffix Template: pSB3K3 PCR3. Primer forward: Suffix Primere reverse: Bluepromoter-Preffix Template: pSB1T3 PCR4. Primer forward: Suffix Primere reverse: OmpFpart1-Preffix Template: pSB1T3 General PCR mixture: 4 microL Template(dilution 1:4) 3 microL Primer forward (5mM) 3 microL Primere reverse (5mM) 6 microL HPLC 6 microL 3.3X XL Buffer 3 microL Mg solution 5 microL dNTP's (0.4 mM) Enzyme(RTTH) mixture for PCR Reaction: 10.5 microL HPLC 9 microL 3.3X XL Buffer 0.5 microL RTTH Program [35 PCR cycles] 94ºC 5min 94ºC 45seg 60ºC-55ºC 45seg 72ºC 10min 4ºC 3min