Monday, August 29th
Agenda:
Tasks:
Tasfia
- Made 5 mL overnight cultures of "TetR-Cas9," "Assembled Cas9 from Cam Culture," GFP from iGEM kit
- Three different colonies
- Used 1000X Cam
- Working concentration was 35 ug/mL
- Added 5.15 uL to each culture tube
- Made 5 mL overnight culture of gRNA Gibson product
- One colony, duplicate overnight cultures
- Used 1000X Tet (10 mg/mL)
- Working concentration was 10 ug/mL
- Added 5 uL to each culture tube
- Gel extracted signal sequences (with homology added)
- Used team-modified protocol:
- Sat product for ~4 minutes between washes
- Evaporated EtOH for ~12 minutes
- Eluted in nuclease-free water after standing for ~15 minutes
- Going forward: we can attempt a Cas9-SS Gibson after figuring out what’s wrong with the gRNA Gibson assembly
Tyler
- Retransformed gRNA Gibson product from 08.26.16
- 3 µL of gRNA Gibson assembly product
- 2 µL positive Gibson kit control
- 2 µL (-) control 1 µL transformation control
- Retransformed 1 µL GFP from iGEM kit
Tasfia
- Made 5 mL overnight cultures of "TetR-Cas9," "Assembled Cas9 from Cam Culture," GFP from iGEM kit
- Three different colonies
- Used 1000X Cam
- Working concentration was 35 ug/mL
- Added 5.15 uL to each culture tube
- Made 5 mL overnight culture of gRNA Gibson product
- One colony, duplicate overnight cultures
- Used 1000X Tet (10 mg/mL)
- Working concentration was 10 ug/mL
- Added 5 uL to each culture tube
- Gel extracted signal sequences (with homology added)
- Used team-modified protocol:
- Sat product for ~4 minutes between washes
- Evaporated EtOH for ~12 minutes
- Eluted in nuclease-free water after standing for ~15 minutes
- Going forward: we can attempt a Cas9-SS Gibson after figuring out what’s wrong with the gRNA Gibson assembly
Tyler
- Retransformed gRNA Gibson product from 08.26.16
- 3 µL of gRNA Gibson assembly product
- 2 µL positive Gibson kit control
- 2 µL (-) control 1 µL transformation control
- Retransformed 1 µL GFP from iGEM kit
