Imperial/Wet Lab/Protocols/CE1.4

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Using the Home-made S30 E. Coli cell extract

Aims

Proper experimental amounts for reaction mixtures of the home-made S30 E.coli cell extract.

Equipment

  • Eppendorf Tubes
  • Gilson p20,p200,p1000

Reagents

  • Pyruvate kinase
  • rNTPs
  • S30 cell extract (home made)
  • Reaction buffer (home made)
  • Commercial S30 cell extract
  • Commercial Pre-incubation mix
  • Amino Acids
    • Minus Cysteine
    • Minus Leucine
  • DNA from midiprep/maxiprep

Protocol

  1. One reaction mixture comprises:
  • Home made S30 - 16.2ul
  • Reaction Buffer- 30ul
  • rNTP's - 1ul
  • Pyruvate Kinase - 3.1ul
  • DNA - 4ul
  • ddH2 - 5.7ul
  1. Then add 4ug/2ug worth of DNA, before topping it up to a total volume of 60ul with nuclease free water.