User:Wilfred J. Poppinga/Notebook/Wilfreds Project/2009/08/11

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Metal sensitive promotors protocol #2 Main project page
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Phosphorylation

  • Mix
    • 1 μL PNK
    • 1 μL T4 DNA Ligase buffer
    • 8 μL Oligo’s (FW and REV seperate reactions)

100x diluted oligo’s (1 μM)

  • Procedure
    • 30 min 37 °C
    • 20 min 65 °C

Annealing

  • Mix
    • 8 μL FW Phosphorylation mix
    • 8 μL REV Phosphorylation mix
    • 4 μL 10 mM NaCl low?
    • 20 μL MilliQ
  • Procedure
    • Put reactions in (close to) boiling water
    • Let it cool down naturally to ~35 °C

Restriction vector

  • Mix
    • 1 μL EcoRI
    • 1 μL XbaI
    • 2 μL Fast digest buffer (10x)
    • 8 μL Vector DNA
    • 8 μL MilliQ
  • Procedure
    • 37 °C, 30 min. incubation
    • Heat inactivation EcoRI and XbaI

5 min. 80 °C 15 min. 65 °C

Ligation of Metal sensitive promoters in vector

  • Mix
    • 2 μL T4 Ligase Buffer
    • 0.5 μL T4 Ligase
    • 1 μL Restriction product (unpurified)
    • 1 μL Annealed oligo’s
    • 18 μL MilliQ
  • Procedure
    • Incubation 16 °C, 1 hour
    • Heat inactivation Ligase

65 °C, 10 min.

Comments

  • Jolanda's ligation mix was transformed
  • Metal sensitive promotors were ligated
    • -RBS according to paul
    • +RBS & -RBS according to my annealing and Paul's restriction ligation



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