User:Jn323/26 June 2012

From 2012.igem.org

Contents

Freeze-Thaw Assay, 1-hour cycles

IPTG Induction
  • Scrape off 4-5 colonies of transformed origami cells and untransformed origami cells from respective plates cultured overnight
  • 3 vials- #1: transformed origami cells, #2: untransformed origami cells, #3: untransformed origami cells
  • Place into 2mL LB media with 2uL Kanomycin stock soln (final concentration of antibiotic: 50ug/mL)
  • Grow for 3 hours in incubator with shaking at 37°C
  • Add 1uL of 0.4M IPTG
  • Added to two of the vials, one of transformed origami and one of untransformed origami
  • Grow for another 3 hours
Freeze-Thaw Test
  • Take concentrations of cells
  • RESULTS - OD600: Transformed Origami exposed to IPTG (TransOri) - 0.27, Untransformed Origami exposed to IPTG (N.iptg) - 0.52, Untransformed Origami unexposed to IPTG(NormOri) - 0.47
  • Aliquot TransOri, N.iptg, and NormOri into 15mL falcon tubes as follows-
  • NOTE - as a way to compensate for lower concentration of TransOri (by approximately a factor of 1/2), double the volume of TransOri culture was added
Tube #1 Tube #2 Tube #3 Tube #4 Tube #5 Tube #6 Tube #7 Tube #8 Tube #9
TransOri 200uL 0uL 0uL 180uL 180uL 100uL 100uL 20uL 20uL
N.iptg 0uL 100uL 0uL 10uL 0uL 50uL 0uL 90uL 0uL
NormOri 0uL 0uL 100uL 0uL 10uL 0uL 50uL 0uL 90uL
  • Add LB media to each falcon tube to reach 3mL mark
  • Lower volume used to increase concentration of cells in solution
  • Place rack in -20°C freezer
  • Remove rack of test tubes from freezer after 1 hour
  • Thaw solution for 30 minutes
  • Plate each test tube of cells onto Kan plates
  • Place rack of test tubes back into freezer for another hour after a full hour has elapsed from time of removing rack from the freezer
  • This cycle is repeated for seven cycles (14 hours total)
  • This test was done overnight from 6:00pm to 8:00am
Results

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