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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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