User:Daniela A. Garcia S./Notebook/Modeling UNAM-Genomics Mexico/2010/08/17

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Wet Lab: OmpC/F promoter

OmpC digestion

The OmpCp is inserted in the plasmid pSB1A2 between the EcoRI and XbaI restriction enzymes. It is done a digestion process to validate the two sites for the restriction enzymes.

  • DNA --------------------20μL
  • BSA --------------------0.3μL
  • Buffer 4 ----------------3μL
  • EcoRI enzyme ---- 1μL
  • XbaI enzyme --------1μL
  • HPLC -------------------4.7μL

Total 30μL

Overnigth reaction @ 37ºC

OmpF RTTH PCR

PCR1

  • FWD Suffix
  • REV OmpF (2)

Template: PCR4(2010/08/05)


PCR2

  • FWD Preffix
  • REV Suffix

Template: PCR5(2010/08/05)


  • 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:
    • 21 μL HPLC
    • 18 μL 3.3X XL Buffer
    • 1 μL RTTH
      • 20 μL for each reaction


  • Program [35 PCR cycles]
    • 94ºC 5min
      • 94ºC 45seg
      • 55ºC 45seg
      • 72ºC 3min
    • 72ºC 10min
    • 4ºC 3min

Hold

  • C2R1S1
  • C3R1S3