August, 1st
For each plate from the previous day, two colonies were picked.
Next, MGZ1 competent cells were prepared according to protocol. They were put in 22 100 μl test-tubes.
August, 2nd
Plasmids containing E28AGAIN-1, E28AGAIN-2, E41AGAIN-1, E28AGAIN-2, E37-2, E38-1. E39-1, E40-2 and E42-1 were extracted with MiniPrep kit from ON saturated cultures; purified DNA was quantified with NanoDrop Spectrophotometer:
| Plasmid |
DNA (ng/μl) |
| E28AGAIN-1 |
21.4 |
| E28AGAIN-2 |
21.0 |
| E41AGAIN-1 |
33.7 |
| E41AGAIN-2 |
27.7 |
| E37-2 |
18.5 |
| E38-1 |
26.0 |
| E39-1 |
21.6 |
| E40-2 |
19.9 |
| E42-1 |
25.1 |
J101-31, J101-E5, J101-E7 were transformed in MGZ1 competent cells.
Digestions of E28AGAIN-1, E28AGAIN-2, E41AGAIN-1 and E42AGAIN-2 were performed for screening; a 5x mix was prepared and for each sample the volumes specified in the table below were used:
| DNA (μl) |
H2O (μl) |
Enzyme 1 (μl) |
Enzyme 2 (μl) |
Buffer H (μl) |
Final Volume (μl) |
| 5 |
16.5 |
0.5 EcoRI |
0.5 Pstl |
2.5 |
25 |
TOP10 competent cells, harboring BBa_T9002 construct, were co-transformed with ENTERO4C5 to confer resistance to Cloramphenicol (12.5 mg/ml).
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