IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/07/25

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Results from Yesterday

  • Kan resistance of biobricks
Biobrick Antibiotic Contration (μg/mL) Observations Conclusion
I746001 Kan 25 Many colonies Kan Resistance OK
I746101 Kan 25 Many colonies Kan Resistance OK
Control PUC19 Kan 25 No colony No Kan Resistance


  • Antibiotic test

Results for Amp

Even with a concentration of 10μg/mL, Amp kills Bacillus. Since we want to know which is the lowest concentration of Amp which kills B.S, we are going to test with some lower concentrations.

Results for Cm

Even with a concentration of 35μg/mL, ther is not a clear area around the antibiotic disk. So we have to test some higher concentration of Cm.


Wet Work

  • New antibiotic tests

Dilution of Amp

Concentration (μg/mL) 10 7.5 5 2.5 1
10 μg/mL Stock (from yesterday) 1:1 3:4 1:2 1:4 1:10


Dilution of Cm

Concentration (μg/mL) 35 37.5 40 42.5 45
35 μg/mL Stock (from yesterday) 1:1 5:6 (from 45μg/mL) 1:875 17:18 (from 45μg/mL) 9:7000

- Melt Soft Agar - 3mL SA + 10μL Cells 1A1 from LB prepared on 23/7/08 - Pour SA+Cells over blank hard agar plates - Put 5 blank disks on each agar plate - Add 40μL of antibiotic on each disk - Incubate at 37°C

Results

Nothing! 1A1 cells were kept in the freedge! B.S. can not be kept in the fridge, low temperatures kill them!


  • Test if biobrick of last year are really the one we want

- Yesterday : Single colony of I746001 and I746101 transferred in 10mL of LB + Amp (final concentration 100μg/mL) - Take 8mL of I746001 (AIP sender) and of I746101 - Centrifuge them - Saved 2ml of each in tubes to regrow and add LB - Miniprep I746001 and I746101 pellets (plasmid mniprep kit) : same protocol but 60μL of Zyppy Glution Buffer in step 8

- Restriction Digest : use EcoI and Spel restriction enzymes - Add sequentially : 15μL of SDW + 2μL of 10X Fast Digest Buffer + 2μL of DNA + 1μL of EcoI + 1μL of Spel - Microfuge tubes - Incubate for 10min at 37°C - Heat inactivation of enzymes at 80°C for 5min

- GEL 20μLof samples + 4μL of dye


  • Grow 1A1

- 10mL of LB + 15μL of 1A1 - Grow at 37°C


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