IGEM:IMPERIAL/2007/Experimental Design/Phase1/Protocol 3.2
- Fluorometer + PC
- 1 well plate
- Plate sticker lid
- Pyruvate kinase
- 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
- 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
- Fill a second eppendorf tube with two samples (40ul x 2) of commercial cell extract including the amino acids mixture, and preincubation buffer.
- Place a quarter of each mixture in a well in the 96 well plate, making up 2 wells (B3 and B5).
- In another well (C4), as a control, place another quarter of only the commercial cell extract, with 28ul of water.
- In the last well (D5), add a quarter of each mixture. This serves as the negative control.
- In the last well, add nuclease free water (again as a negative control).
- In wells B3, B5 and C4, add 20ul of DNA.
- Measure the fluorescence at 30 min intervals for the 1st hour, and hourly intervals thereafter.
- After each measurement, cover the plate with the sticky lid.