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






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