IGEM:IMPERIAL/2007/Experimental Design/Phase1/Protocol 3.2

Equipments

• Fluorometer + PC
• 1 well plate
• Plate sticker lid

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
• pTet DNA plasmid

Steps

1. Fill an eppendorf tube with two samples (56ul x 2) of home made cell extract including reaction buffer, pyruvate kinase and rNTPs.The volumes are shown below:
   • Home made S30 - 16.2ul
   • Reaction Buffer- 30ul
   • rNTP's - 1ul
   • Pyruvate Kinase - 3.1ul
   • DNA - 4ul
   • ddH₂ - 5.7ul
2. Fill a second eppendorf tube with two samples (40ul x 2) of commercial cell extract including the amino acids mixture, and preincubation buffer.
3. Place a quarter of each mixture in a well in the 96 well plate, making up 2 wells (B3 and B5).
4. In another well (C4), as a control, place another quarter of only the commercial cell extract, with 28ul of water.
5. In the last well (D5), add a quarter of each mixture. This serves as the negative control.
6. In the last well, add nuclease free water (again as a negative control).
7. In wells B3, B5 and C4, add 20ul of DNA.
8. Measure the fluorescence at 30 min intervals for the 1st hour, and hourly intervals thereafter.
9. After each measurement, cover the plate with the sticky lid.