User:Christine Doan/Notebook/Identifying Kinase Substrates of PfCK2/2014/03/11

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new CK2

  • RIPL plate did not grow
  • Try transformation again into BL21 RIPL and RIL to compare
  • Andrew is making new Amp plates

Substrates

  • Subs 2 and 9 have DNA concentrations too low to clone
  • Ran cleanups on gel, look good
  • Can re-PCR to amplify >70 and use those products for cloning into pET-30
    • 50ul GoTaq PCR rection
      • 25ul GoTaq Master Mix
      • 1ul forward primer
      • 1ul reverse primer
      • 1-5ul DNA template (depending on what kind of DNA, concentration, end goal, etc)
      • Nuclease-Free Water up to 50ul
    • PCR
      • 95C 2mins denature
      • 95C 30sec denature
      • 39C 1min anneal
      • 68C 5min extend
      • 68C 10min extend
      • 4C hold
  • T4 treatment for cloning requires 0.2 picamole