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(Infinite F200 readings)
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**485/20 X3
**485/20 - we have three of this

Revision as of 10:00, 28 March 2013

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Infinite F200 readings

The microplate reader can be used for static or dynamic readings of bacteria cultures growth and fluorescent proteins.

Static readings

  • Grow bacteria under the desired conditions.
  • Transfer 200 ul of culture in a well of a 96-well microplate.
  • Measure with or without lid.
  • Always measure a "blank" that can be subtracted to the other raw measurements (blank for absorbance is sterile media; blank for absorbance is non-fluorescent culture grown to an OD600 comparable to the fluorescent cultures).

Dynamic readings

  • Grow bacteria under the desired conditions.
  • Transfer 200 ul of culture in a well of a 96-well microplate, excluding the 36 wells of the "frame".
  • Dispense deionized water in all the unused wells.
  • Add inducers to the cultures when required.
  • Start a kinetic cycle of 18 hours with the i-control software (Tecan): 37degC during all the experiment, linear shaking (15 s, 3 mm amplitude), 5 s wait time (also 10 s is ok), absorbance reading (default parameters), fluorescence readings (gain=50 for medium and high copy plasmids, gain=50 or 80 for low copy plasmids, gain=80 or 100 for single-copy integrants), sampling time=5 min. The specified fluorescence gain factors are only guidelines and can be modified according to promoter/RBS strength, strain, growth medium and cell density. RFP filters are 535/620nm, GFP filters are 485/540nm.
  • Always include a "blank" for absorbance and fluorescence, as described before.
  • The microplate should be covered with lid for dynamic readings >5-6 hours.

List of filters

  • for fluorescence (nm/bandwidth):
    • 620/20
    • 560/20
    • 540/25
    • 535/25
    • 485/20 - we have three of this
    • 430/35
  • for absorbance (nm):
    • 600
    • 340
  • Commonly used filter pairs for fluorescent proteins (EX=excitation filter, EM=emission filter):
    • CFP EX:430 EM:485
    • GFP EX:485 EM:540
    • YFP EX:485 EM:560
    • RFP EX:535 EM:620