IGEM:Imperial/2007/Wet Lab/Protocols/Prot1.9

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 Turn on before beginning
• 96 well plate x1 + Plate lid
• 1.5ml eppendorf tube x7
• eppendorf rack
• Gilson p20,p200,p1000
• Stop watch

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

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)

1. Place the plate in the fluorometer to measure its initial fluorescent reading.
2. After the measurement, place the sticky tape across the plate, and put the plate in the 8oC water bath.
3. Start on the next plate, and repeat procedures 2-6.
4. Place the plate in the 15oC water bath.
5. Repeat the same procedures for the remaining two plates, except that before placing them in the water bath, wrap aluminium foil around them to prevent photobleaching. (This is because the water baths 25oC and 30oC will be out in the openand exposed to light)
6. Stagger the start of all the plates by around 5 minutes, and measure the temperature every 30 minutes for each temperature.
7. Continue this for 6 hours, and plot a graph of the fluorescence against time for all 4 plates.