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Team:UNIPV-Pavia/Notebook/Week17
From 2008.igem.org
(Difference between revisions)
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*We prepared 0.5 l of LB + Amp for liquid cultures. | *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 | + | *We infected 9 ml of LB + Amp with 30 µl of Lig.30(3), Lig.31, Lig.22, Lig.13 and two falcon tubes for Lig.a. |
*We received 3OC6HSL from Sigma! we resuspended it in ddH2O, we prepared some stocks and stored them at -20°C. | *We received 3OC6HSL from Sigma! we resuspended it in ddH2O, we prepared some stocks and stored them at -20°C. | ||
| Line 62: | Line 62: | ||
'''09/9/08''' | '''09/9/08''' | ||
<br> | <br> | ||
| - | *Glycerol stocks/miniprep for 30(3), 31, 22 and 13. | + | *Glycerol stocks/miniprep for Lig.30(3), Lig.31, Lig.22 and Lig.13. |
*Plasmid digestion for: | *Plasmid digestion for: | ||
| - | **30 (S-P) | + | **Lig.30 (S-P) |
| - | **31 (S-P) | + | **Lig.31 (S-P) |
| - | **30 (X-P) | + | **Lig.30 (X-P) |
| - | **22 (X-P) | + | **Lig.22 (X-P) |
**13 (X-P) | **13 (X-P) | ||
| Line 74: | Line 74: | ||
*Ligations: | *Ligations: | ||
| - | **31-30 (="34") | + | **Lig.31-Lig.30 (="Lig.34") |
| - | **31-22 (="35") | + | **Lig.31-Lig.22 (="Lig.35") |
| - | **30-13 (for green fluorescence test) | + | **Lig.30-Lig.13 (="Lig.T5" for green fluorescence test) |
| - | *We induced one of the two | + | *We induced one of the two Lig.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: | *Fluorescence test results: | ||
| Line 93: | Line 93: | ||
*Plates grew correctly and diluted transformation showed less colonies. | *Plates grew correctly and diluted transformation showed less colonies. | ||
| - | *Colony PCR for 34, 35 and | + | *Colony PCR for Lig.34, Lig.35 and Lig.T5 (4 colonies for normal transformation plates and 4 colonies for diluted transformation plates). |
*Gel results: | *Gel results: | ||
Revision as of 17:53, 2 October 2008
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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 Lig.30(3), Lig.31, Lig.22, Lig.13 and two falcon tubes for Lig.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 Lig.30(3), Lig.31, Lig.22 and Lig.13.
- Plasmid digestion for:
- Lig.30 (S-P)
- Lig.31 (S-P)
- Lig.30 (X-P)
- Lig.22 (X-P)
- 13 (X-P)
- Run/gel extraction.
- Ligations:
- Lig.31-Lig.30 (="Lig.34")
- Lig.31-Lig.22 (="Lig.35")
- Lig.30-Lig.13 (="Lig.T5" for green fluorescence test)
- We induced one of the two Lig.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 Lig.34, Lig.35 and Lig.T5 (4 colonies for normal transformation plates and 4 colonies for diluted transformation plates).
- Gel results:
