"
Team:Paris Liliane Bettencourt/Notebook/2011/08/13/
From 2011.igem.org
(→Adding the RFP biobrick in pHM3) |
|||
| Line 82: | Line 82: | ||
It is incubated one hour at 22°C with some mixing from time to time, and then the totality of the product will be transformed in MH1 cells | It is incubated one hour at 22°C with some mixing from time to time, and then the totality of the product will be transformed in MH1 cells | ||
| + | |||
| + | == Kevin == | ||
| + | ===Results of transformation === | ||
| + | |||
| + | All the plates has grown with uniform cultur and no spots. | ||
| + | Positive control (S27) are fluorescent, and not all the others, so digestion works. | ||
| + | |||
| + | We have to test if the transformation works. | ||
| + | |||
| + | ===Cultur of transformated cells to test=== | ||
| + | Cultur overnight from cells in the plate has been done. | ||
Revision as of 15:20, 15 August 2011
Contents |
Cyrille
Adding the RFP biobrick in pHM3
We want to insert the biobrick J04450 into pHM3 for three reasons:
- To have a negative control of digestion
- To restore the EXSP biobrick format in the plasmid
- To test the possibility of cloning in this plasmid
3 times 500 ng of the pHM3 plasmid is digested in E P for 30 min
3 times 500ng of J04450 in pSB1C3 is digested using E P sites.
The result is loaded on a gel. The and will undergo a gel purification.
After the gel extraction the yields where very low, so that it was in the noise of the tecan machine. So I decided to pool together the threee tubes of the same serie. Once mixed, vortexed and centrifucated, the concentration was mesures again. The tecan says the concentration is around 10 and 20 ng/mL depending on the tube and on the measure. This is sufficient to carry on.
The ligation was done using the T4 DNA ligase. 5 tubes where prepared as explain:
| Tube | 1 | 2 | 3 | 4 |
| 10x T4 buffer | 2µL | 2µL | 2µL | 2µL |
| T4 DNA ligase | 1µL | 1µL | 1µL | 1µL |
| DNA pHM3 EcoRI- | 2µL | 2µL | 5µL | 5µL |
| J04450 | 2µL | 10µL | 5µL | 10µL |
| H2O | 13µL | 5µL | 7µL | 2µL |
And the control
| 10x T4 buffer | 2µL |
| T4 ligase | 5 µL |
| DNA pHM3 | 5µL |
| H2O | 12µL |
It is incubated one hour at 22°C with some mixing from time to time, and then the totality of the product will be transformed in MH1 cells
Kevin
Results of transformation
All the plates has grown with uniform cultur and no spots. Positive control (S27) are fluorescent, and not all the others, so digestion works.
We have to test if the transformation works.
Cultur of transformated cells to test
Cultur overnight from cells in the plate has been done.
