1. Separate transformant colonies (transformation from previous day) inoculated to liquid LB with kanamycin.
2. Inoculation of pACYC177+OmpA_alpha and pACYC177+OmpA_omega - liquid LB with kanamycin.
Piotr:
Checking if OmpA_omega_A_alpha gives ampicillin resistance.
Inoculation OmpA_omega_A_alpha from various IPTG concentrations (0, 0.1, 0.25, 0.5, 0.75, 1) into the same IPTG concentrations, but with various ampicillin concentrations(25, 50, 75, 100) in relation: 1:50.
Measurement of bacterial culture growth (OD) in the evening:
| ampicillin concentration (mikrogramy na mililitr): | IPTG concentration (milimole na mililitr): |
|---|
| 0 | 0,1 | 0,25 | 0,5 | 0,75 | 1 |
|---|
| 25 | 1,558 | 1,469 | 1,587 | 1,49 | 1,566 | 1,311 |
|---|
| 50 | 1,425 | 1,435 | 1,524 | 1,055 | 0,920 | 0,935 |
|---|
| 75 | 1.09 | 0,989 | 1.447 | 0.971 | 0.951 | 0.992 |
|---|
| 100 | 0.09 | 0,685 | 1.378 | 1.078 | 0.977 | 0.992 |
|---|
Inoculation of OmpA_omega_A_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75 (repeat is needed!!!)
Checking OmpA_omega_A_alpha and OmpA_A_alpha expression.
Spinnig, suspension, adding lysing buffer (INGREDIENTS), boiling, electrophoresis in polyacrylamide gel, Western blot (antibodies anti-A, rinsing,anti-rabbit secondary antibodies , processing with BCIP and NBT (OmpA_omega_A_alpha with and without induction, OmpA_A_alpha)
(Zdjęcie żelu)
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