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Team:UNIPV-Pavia/Notebook/Week17
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Notebook
| Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 |
|---|---|---|---|---|---|---|
| Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 |
| Week 15 | Week 16 | Week 17 | Week 18 | Week 19 | Week 20 | Week 21 |
Week 16: 09/8/08 - 09/12/08
09/8/08
- We prepared 0.5 l of LB + Amp for liquid cultures.
- We infected 9 ml of LB + Amp with 30 µl of 30(3), 31, 22, 13 and two falcon tubes for "a".
- We received 3OC6HSL from Sigma! we resuspended it in ddH2O, we prepared some stocks and stored them at -20°C.
09/9/08
- Glycerol stocks/miniprep for 30(3), 31, 22 and 13.
- Plasmid digestion for:
- 30 (S-P)
- 31 (S-P)
- 30 (X-P)
- 22 (X-P)
- 13 (X-P)
- Run/gel extraction.
- Ligations:
- 31-30 (="34")
- 31-22 (="35")
- 30-13 (for green fluorescence test)
- We induced one of the two "a" overnight culture with 3OC6HSL 1 µM. We incubated the two cultures for 1 hour and then watched TRITC channel at microscope. (We didn't synchronize the two cultures, but performed a qualitative test for luxR mutated protein integrity evaluation).
- Fluorescence test results:
09/10/08
- We transformed/plated ligations. We decided to perform two transformations for each ligation:
- one normal transformation (1 µl of ligation);
- one diluted ligation (1:10)
- We decided to try diluted transformations to have less colonies in the plate and to avoid streaking single colonies plates when colonies are not insulated.
09/11/08
- Plates grew correctly and diluted transformation showed less colonies.
- Colony PCR for 34, 35 and 30-13 (4 colonies for normal transformation plates and 4 colonies for diluted transformation plates).
- Gel results:
