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Team:Chiba/Notebook/Calendar/24 September 2009
From 2009.igem.org
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== To judge character of LuxR mutants(3)-1 == | == To judge character of LuxR mutants(3)-1 == | ||
| + | We poured 1 mL of LB-Amp, Cm liquid medium in 96 deep well and added glycerol stocks. | ||
| + | |||
| + | |||
22:10- | 22:10- | ||
| + | |||
| + | We cultured and shook it at 37 degrees Celsius. | ||
Revision as of 16:36, 25 September 2009
(23_September_2009 <|>25_September_2009)
Contents |
Examine limit of AHL generation
Yesterday's operation is here.
- Today's operation
14:00
We made plates from each of mixture and 10 mL of LB-Amp, Cm solution medium.
- Element of mixtures
| Sample Namber | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| supernatant solution | 5 mL | --- | --- | --- | --- | --- | --- | 20 μL |
| 1 | --- | 5 mL | --- | --- | --- | --- | --- | --- |
| 2 | --- | --- | 5 mL | --- | --- | --- | --- | --- |
| 3 | --- | --- | --- | 5 mL | --- | --- | --- | --- |
| 4 | --- | --- | --- | --- | 5 mL | --- | --- | --- |
| 5 | --- | --- | --- | --- | --- | 5 mL | --- | --- |
| 6 | --- | --- | --- | --- | --- | --- | 5 mL | --- |
| LB-Amp,Cm | 10 mL | 10 mL | 10 mL | 10 mL | 10 mL | 10 mL | 10 mL | 9.8 mL |
| Total | 10 mL | 10 mL | 10 mL | 10 mL | 10 mL | 10 mL | 10 mL | 10 mL |
Transformation(2)-2
Yesterday's operation is here.
- Today's operation
11:15-
We picked colony(plux-GFP only) and cultured it.
15:50-
Main culture
20:30-
Mini prep.
And resultant DNA was saved in freezer.
Transformation(3)-1
- Plasmids
ptet-GFP pSB1A2 [http://partsregistry.org/Part:BBa_I13522 BBa_I13522](Amp)
ptet-RFP pSB1A3 [http://partsregistry.org/Part:BBa_I13521 BBa_I13521](Amp)
ptet-CFP pSB1A2 [http://partsregistry.org/Part:BBa_I13600 BBa_I13600](Amp)
mRFP without Nco1 site(Cm)
- Competent Cells
21:50-
We cultured each plates.
Digestion Test
- Samples
1 : ECFP([http://partsregistry.org/Part:BBa_I6057 BBa_I6057])
2 : mCherry([http://partsregistry.org/Part:BBa_E2060 BBa_E2060])
3 : V-YFP([http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
4 : mOrange([http://partsregistry.org/Part:BBa_E2050 BBa_E2050])
5 : LacZ α([http://partsregistry.org/Part:BBa_T9003 BBa_T9003])
6 : mRFP without Nco1 site
- Doble Digestion
| Sample | 3 μL |
| EcoR1 | 0.20 μL |
| Pst1 | 0.20 μL |
| Buffer | 1 μL |
| BSA | 1 μL |
| dw | 4.6 μL |
| Total | 10 μL |
- Doble Digestion's Master Mix
| Sample | 3 μL |
| EcoR1 | 1.4 μL |
| Pst1 | 1.4 μL |
| Buffer | 7 μL |
| BSA | 7 μL |
| dw | 32.2 μL |
| Total | 49 μL |
- Single Digestion's Master Mix
| EcoR1 | 1.4 μL |
| Buffer | 7 μL |
| dw | 40.6 μL |
| Total | 49 μL |
- Doble Digestion
| Sample | 3 μL |
| EcoR1 | 0.20 μL |
| Pst1 | 0.20 μL |
| Buffer | 1 μL |
| BSA | 1 μL |
| dw | 4.6 μL |
| Total | 10 μL |
12:50
Warm up at 37 degrees Celsius
13:30
Gel electrophoretic analysis
To judge character of LuxR mutants(3)-1
We poured 1 mL of LB-Amp, Cm liquid medium in 96 deep well and added glycerol stocks.
22:10-
We cultured and shook it at 37 degrees Celsius.
