Template:IONIS Paris/Notebook/16-07-15
Results of bacterial transformation
No colonies
We should try again with a plasmid we already transformed
Bacterial survival has to be tested
Digestion of Gibson product (VVD-YC155)
NEB double digestion: Buffer 2.1
EcoRI:100%, PstI:75%
| Tube | Gibson | Gibson |
|---|---|---|
| Water | 12 µL | 11,5 µL |
| Buffer 2.1 | 2 µL | / |
| Buffer 3.1 | / | 2 µL |
| Plasmid | 5 µL | 5 µL |
| Enzyme EcoRI | 0,5 µL | 0,5 µL |
| Enzyme PstI | 0,5 µL | 0,5 µL |
| Enzyme EcoRV | / | 0,5 µL |
37°C, 100 min
Electrophoresis of digested products
Expected results
Results
We get confirmation of the presence of pSB1C3 and our part, but there is still the unknown band. We have cut the heaviest band of the fourth wells to isolate our plasmid from the other one.
Test of competent cells: transformation with pSB1C3-RFP
Transformation of 100µl of homemade competent cells with 1 µl of pSB1C3 containing a RFP following our protocol and plated onto two agar plates containing chloramphenicol
100 µl of homemade competent cells have been split and plated onto two agar plates, one with chloramphenicol and the other one without antibiotic
16 July 15