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Protocols for CBD temperature step experiment

Aims:

• To determine the behaviour of Cell By Date in situations of temperature step-up or step-down.
• We will be carrying out 4 experiments under the following incubation conditions:
  • 4°C for 6h
  • 20°C for 6h
  • 4°C for 3h and 20°C for next3h
  • 20°C for 3h and 20°C for another 3h

Equipment

• Fluorometer + Connected PC
• Water bath in cold room at 20°C
• 4 Fluorometer plates (black)
• Gilson pipettes p200 p20 p10
• Eppendorf Tubes
• Stopwatch

Reagents

• 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 pTet-GFP from maxiprep

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 20°C.
3. Commercial E.coli Cell Extract: First prepare cell extract for 18 reactions. Add 45μl each of two amino acid minus mixtures into an eppendorf tube. This gives a total volume of 90μl. 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. To the same eppendorf tube, add 360µl of S30 Premix. Then add 270µl of S30 Extract Circular.
4. DNA constructs We need to prepare a dilution of DNA to give 4ug of DNA. Add 104ul of pTet-GFP DNA to 156ul of Nuclease Free water (13 reactions worth based on [DNA] = 500ng/ul). Add 60.5ul of empty vector DNA to 39.5ul of Nuclease Free Water (5 reactions worth based on [DNA] = 330ng/ul).

Loading Plate

1. Follow the schematic for the plate and begin by loading the in vitro expression system into the correct wells. Before loading in the samples vortex the tubes for a few seconds to mix the solution.
2. Tap down the top of the lid to bring down any solution to bottom of the well.
3. Remove lid off the 96 well plate and place in the fluorometer. Create a file name insert temp under: D:\IGEM\INSERT DATE\CBD. Export the data here. Each file should be named as the following:
   • construct-temp-time-date
4. This measurement will give a back ground fluorescence measurement and can be used as our time zero data.
5. Then to begin the reaction add 4ug of purified DNA sample to each well indicated on the schematic.
6. Place the plate in the fluorometer to measure its initial fluorescent reading.
7. After the measurement, place the sticky tape across the plate, and put the plate in the 20oC water bath.
8. Start on the next plate, and place it in the 4oC cold room.
9. Before placing them in the water bath, wrap aluminium foil around them to prevent photobleaching.
10. Stagger the start of all the plates by around 5 minutes
11. Measure the temperature every 30 minutes for each temperature and repeat for 6 hours.