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From 2012.igem.org

Contents 1 Freeze-Thaw Assay, 1-hour cycles 1.1 IPTG Induction 1.2 Freeze-Thaw Test 1.3 Results

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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