From 2007.igem.org

Testing pT7-GFPmut3b in-vitro - Protocols

Aims:

• To determine if pT7- GFP DNA constructs for Infector Detector expresses in vitro
• We will be testing at the following temperature: 4 °C, 25°C, 37°C

Equipment

• Fluorometer + Connected PC
• 3 Fluorometer Plates (Black)
• Sticky Seal Tape
• Eppendorf Tubes
• Gilson p20,p200,p1000
• Stop watch
• Foil

Reagents

• Our Prepared S30 extract
• Commercial S30 E.coli extract. Including:
  • 175µl Amino Acid Mixture Minus Cysteine, 1mM
  • 175µl Amino Acid Mixture Minus Methionine, 1mM
  • 175µl Amino Acid Mixture Minus Leucine, 1mM
  • 450µl S30 Extract, Circular (3 × 150µl)
  • 750µl S30 Premix Without Amino Acids
• Nuclease Free water x1ml
• DNA pT7-GFP from midiprep
• DNA pT7 from midiprep

Protocols

1. First collect all equipment and reagents and ensure that the fluorometer and that the PC connected has a data collection protocol installed.
2. Turn on the water bath at 25 °C and 37 °C incubator.
3. Commercial E.coli Cell Extract: First prepare cell extract for 13 reaction. Add the 32.5μl volume of two amino acid minus mixtures into an labeled eppendorf to give a volume of 66μl. Each amino acid minus mixture is missing one type of amino acid, and so by combining two solutions we are complementing each solution for the missing amino acid. Place eppendorf in a rack on bench. Take an eppendorf tube and add 260µl of S30 Premix. Then add 195µl of S30 Extract Circular.
4. DNA constructs We need to prepare a dilution of DNA to give 4ug of DNA. Add 64 of pT7-GFP DNA to 136ul of Nuclease Free water (10 reactions worth). Add 39.2 ul of pT7 DNA to 20.8ul of Nuclease Free Water (4 reactions worth)

Loading Plate

1. To each of the plate, place the cell extracts in 4 wells, and add the pT7-GFP to 3 of the wells, and pT7 DNA to the last well. Then top up each well to a total volume of 60ul.
2. Place the plate in the fluorometer to measure its initial fluorescent reading.
3. After the measurement, place the sticky tape across the plate, and put the plate in the 37oC water bath.
4. Start on the next plate, and place the plate in the 25oC water bath.
5. Repeat the same procedures for the remaining plate at 4oC
6. Before placing them in the water bath, wrap aluminium foil around them to prevent photobleaching.
7. Stagger the start of all the plates by around 5 minutes.
8. Repeat the measurements every hour, for 6 hours.