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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.