Wednesday, July 20th
Agenda:
- Meeting with Traci
- Read the Golden Gate stuff from Patrick
- Call millipore
- Gibson of Cas9 parts and tet backbone
- Transform the Gibson products
- Miniprep
- Sequencing
- Linearize
- Meet with Patrick
- gRNA construct
- Look into Weinberg and Provost travel grants
- Fun antibiotic activity design
Tasks:
Jordan
- Read GG protocol papers
- Met with Traci from iGEM and UChicago
Sam
- Continued compiling list of experts
- Met with Prof. Broadbelt
- Ran transformation (no controls):
- Thawed 100 uL of comp cells
- Added 5 uL of Gibson results to the comp cells
- 15 uL of Gibson left over
- Will run a gel with a little of it tomorrow
- Mix sat 30 min on ice
- 45 sec in water bath at 42°C
- 5 min back on ice
- 900 uL SOC broth added under flame
- Shaking incubator at 37°C for 1 hr
- Pipetted 50 uL and 100 uL onto 2 (total) Cam plates and let grow overnight
- Plates were a little on the cold side when we plated
- Gel:
- 50 mL recycled gel
- 5 uL SybrGreen
- Did not cover while solidifying. Not used to covering because SyberSafe isn’t as sensitive
Sara
- Patrick showed us how to phosphorylate the ends of the primer dimers/make them anneal to each other in prep for Golden Gate
- Peter helped us run through the Gibson
- Resuspended Cas9(1) and Cas9(2) in 10nL of nuclease free water, bring concentration down to 100 ng/uL
- Gibson assembly
Shu
- Resuspended Cas9 parts in water with Sara
- Used 10 uL of the Gibson master mix
- 6.25 uL NF water
- 0.7 uL linearized Tet backbone
- 1.6 uL Cas9(1)
- 1.45 uL Cas9(2)
- 1 hr at 50°C
- Total of 20 uL
- Learned Golden Gate with Patrick
- Gibson assembly with Peter
- Golden Gate paper reading
Tasfia
- Gibson assembly (see Sara’s entry)
- Started transformation (see Sam’s notes about transformation)
- Looked into experts for human practices
- Transform the Gibson products
- Miniprep
- Sequencing
- Linearize
Jordan
- Read GG protocol papers
- Met with Traci from iGEM and UChicago
Sam
- Continued compiling list of experts
- Met with Prof. Broadbelt
- Ran transformation (no controls):
- Thawed 100 uL of comp cells
- Added 5 uL of Gibson results to the comp cells
- 15 uL of Gibson left over
- Will run a gel with a little of it tomorrow
- Mix sat 30 min on ice
- 45 sec in water bath at 42°C
- 5 min back on ice
- 900 uL SOC broth added under flame
- Shaking incubator at 37°C for 1 hr
- Pipetted 50 uL and 100 uL onto 2 (total) Cam plates and let grow overnight
- Plates were a little on the cold side when we plated
- Gel:
- 50 mL recycled gel
- 5 uL SybrGreen
- Did not cover while solidifying. Not used to covering because SyberSafe isn’t as sensitive
Sara
- Patrick showed us how to phosphorylate the ends of the primer dimers/make them anneal to each other in prep for Golden Gate
- Peter helped us run through the Gibson
- Resuspended Cas9(1) and Cas9(2) in 10nL of nuclease free water, bring concentration down to 100 ng/uL
- Gibson assembly
Shu
- Resuspended Cas9 parts in water with Sara
- Used 10 uL of the Gibson master mix
- 6.25 uL NF water
- 0.7 uL linearized Tet backbone
- 1.6 uL Cas9(1)
- 1.45 uL Cas9(2)
- 1 hr at 50°C
- Total of 20 uL
- Learned Golden Gate with Patrick
- Gibson assembly with Peter
- Golden Gate paper reading
Tasfia
- Gibson assembly (see Sara’s entry)
- Started transformation (see Sam’s notes about transformation)
- Looked into experts for human practices
