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