Wednesday, July 27th
Agenda:
- Pour 2 gels for GFP and mCherry
- PCR linearization of the Tet-Cas9 backbone in preparation for insertion of ClyA or INP/signaling sequences
- PCR Cleanup of GG gels
Tasks:
Jordan
- Designed ClyA sequencing primers
- Ran gel extraction/cleanup on mCherry’s (Shu did GFPs), followed kit procedure, used 30 ul water not elution buffer, added more buffer up to about 500 ul
- Looked into more fractionation procedures Ligation
Michelle
- Made glycerol stocks of A1–A3, A5, B1–B3, B5 Cas9 cultures
- 500 μL overnight culture
- 500 μL 50% glycerol in dH2O
- In microcentrifuge tube stored in -80°C in Mordacq’s lab, in box marked 2016 iGEM Glycerol Stocks
- Researched Cas9 expression & purification protocols
- Cas9–INP and Cas9–ClyA Gibson math
- Made 5mL cultures of Cas9 from glycerol stocks with Sara
- 50 μL Cam in 5 mL LB
- Cultures A3, A5, B2
- Ran mCherry & sfGFP PCR
- 0.5 μL of 10 μM forward primer
- 0.5 μL of 10 μM reverse primer
- 1 μL template (sfGFP or mCherry miniprep)
- 12.5 μL of OneTaq Master Mix
- 10.5 μL nuclease-free water
- 95°C (2:00) | 95°C (0:07), 51°C (0:10), 72°C (0:43) | 72°C (5:00)
- 25 μL reaction volume, 25 cycles
- Aliquoted 100 mL of LB for autoclaving to grow up Cas9 tomorrow
- Aliquoted LB into smaller bottles for autoclaving tomorrow
Paul
- Looked up Cas9 expression procedure
- Ran gel on mCherry and GFP PCR products
- Helped out with various experiments
Sara
- PCR of Tet-Cas9 backbone with Tyler
- Used DNA from B2 and A5 overnight cultures
- Made 5mL cultures of Cas9 from glycerol stocks with Michelle
- 50 μL Cam in 5 mL LB
- Cultures A3, A5, B2
Shu
- Gel extracted sfGFP for Golden Gate assembly
- Grew mRFP culture
- Looked over SS sequencing primers
Tasfia
- Made glycerol stocks with Michelle
- PCR Cleanup of GFP/mCherry
Jordan
- Designed ClyA sequencing primers
- Ran gel extraction/cleanup on mCherry’s (Shu did GFPs), followed kit procedure, used 30 ul water not elution buffer, added more buffer up to about 500 ul
- Looked into more fractionation procedures Ligation
Michelle
- Made glycerol stocks of A1–A3, A5, B1–B3, B5 Cas9 cultures
- 500 μL overnight culture
- 500 μL 50% glycerol in dH2O
- In microcentrifuge tube stored in -80°C in Mordacq’s lab, in box marked 2016 iGEM Glycerol Stocks
- Researched Cas9 expression & purification protocols
- Cas9–INP and Cas9–ClyA Gibson math
- Made 5mL cultures of Cas9 from glycerol stocks with Sara
- 50 μL Cam in 5 mL LB
- Cultures A3, A5, B2
- Ran mCherry & sfGFP PCR
- 0.5 μL of 10 μM forward primer
- 0.5 μL of 10 μM reverse primer
- 1 μL template (sfGFP or mCherry miniprep)
- 12.5 μL of OneTaq Master Mix
- 10.5 μL nuclease-free water
- 95°C (2:00) | 95°C (0:07), 51°C (0:10), 72°C (0:43) | 72°C (5:00)
- 25 μL reaction volume, 25 cycles
- Aliquoted 100 mL of LB for autoclaving to grow up Cas9 tomorrow
- Aliquoted LB into smaller bottles for autoclaving tomorrow
Paul
- Looked up Cas9 expression procedure
- Ran gel on mCherry and GFP PCR products
- Helped out with various experiments
Sara
- PCR of Tet-Cas9 backbone with Tyler
- Used DNA from B2 and A5 overnight cultures
- Made 5mL cultures of Cas9 from glycerol stocks with Michelle
- 50 μL Cam in 5 mL LB
- Cultures A3, A5, B2
Shu
- Gel extracted sfGFP for Golden Gate assembly
- Grew mRFP culture
- Looked over SS sequencing primers
Tasfia
- Made glycerol stocks with Michelle
- PCR Cleanup of GFP/mCherry
