IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/06/08

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Miniprep of Amp Construction Plasmid and Chlor Construction Plasmid

  • Centrifuged 5mL of each construction plasmids in a 1.5mL centrifuge tube for 4 runs at 14000rpm (Galaxy 16) for 10 min.
  • Add 250uL of Resuspension Buffer with RNAase A and resuspend by vortexing
  • Add 250uL of Lysis Buffer and invert 10 times
  • Inncubate at room temperature for 5 min
  • Add 250uL of Precipitation Buffer and invert 10times
  • Transfer solutions to 2 filter mini columns some precipitate was not transfered because because it was sticking to the tube and pipet
  • Wait 2 minutes
  • Centrifuge at 4100rpm (1350xg) for 1 min
  • Remove Clarification column
  • Add Wash Buffer 1 (750uL) to column

Centrifuge at 10200rpm (8500x g) for 1 min Add 250uL of Wash Buffer 2 and centrifuge at 10200rpm

  • Insert column nto 1.7 elution tube and add 100uL Elution Buffer
  • Spin at 10200rpm for 1 min. Store at 4°C

Results of Miniprep

' ng/ul 260/280 260/230
Amp Construction Plasmid 225.74 2.04 2.59
Chlor Construction Plasmid 149.94 2.00 2.52

NOTE: All results were found using a nanodrop with 1uL of sample

Making Competent BW27783 and DH5α cells

  • Inoculate 5mL overnight culture and grow at 30°C.
  • Dilute 0.5mL overnight culture into 50mL LB and incubate at 30°C, shaking vigorously.
  • Harvest BW27783 at Abs600nm = 0.482 and DH5α at Abs600 = 0.390.
  • Centrifuge at 1600g for 7 minutes (4°C) - (Using AMBL centrifuge, 4500rpm).
  • Wash gently in ~20mL COLD filter sterilized 0.1M CaCl2.
  • Spin down gently: 1100g, 5 minutes, 4°C - (Using AMBL centrifuge, 3700rmp).
  • Resuspend in 12.5mL cold 0.1M CaCl2.
  • Keep on ice 40 minutes.
  • Spin down: 1100g, 5 minutes, 4°C.
  • Resuspend in 2-2.5mL cold 0.1M CaCl2.
  • Store over night at 4°
  • Use 100uL aliquots, store at -80°C?.

Resuspension and Transformation of BioBricks

  1. Worked with Karen and Amelia
  2. Resuspended BioBricks into 15 µL of sdH2O in new PCR tube
Part Description Part # Plate/Well
eYFP E0430 1/8I
RFP E1010 1/18F
J23100 promoter J23100 1/18C
RBS B0034 1/2M