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Revision as of 15:16, 23 October 2007

Contents 1 Protocols for CBD temperature step experiment 1.1 Equipment 1.2 Reagents 1.3 Protocols

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 Turn on before beginning
• 96 well plate x4 + Plate lid
• 1.5ml eppendorf tube x3
• eppendorf rack
• Gilson p20,p200,p1000
• Stop watch

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

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.
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).