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 Template: pSB3K3 + GFP
 * FWD XbaI_RBS_GFP
 * REV OmpF(1)

PCR2 Template: pSB3K3 + GFP
 * FWD XbaI_RBS_GFP
 * REV Bluepromoter-Preffix

PCR3 Template: pSB3K3 + GFP
 * FWD Preffix
 * REV Suffix

> RFP Amplification

PCR4 Template: pSB1C3
 * FWD Suffix
 * REV OmpF (1)

PCR3 Template: pSB1C3
 * FWD Preffix
 * REV Suffix


 * 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


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