Monday, August 15th
Agenda:
- Glycerol stocks of Cas9
- Miniprep Cas9
- Miniprep Tet BB
- PCR Linearize Tet for GFP
- PCR mRFP
- Retransform Cas9-with-SS Gibson
- PCR linearize for ClyA
- Clean out fridge
- PCR linearize mRFP backbone
- DpnI digest
- Run gel and gel extract
- Gibson the gRNA with template
- Transform
Tasks:
Paul
- PCR linearize Tet BB for GFP
- 3 tubes A: (In this order in the gel)
- Original template
- New template
- Half primer concentration (0.25 uL each 10 uM primer)
- 1 tube B: original template
- DpnI digest at 37 C for 2 hours
- Retransform:
- Gibson + control (5 uL)
- Gibson - control (no insert) (3 uL)
- pUC19 (1 uL, 50 pg/uL)
- GG no ligase control (5 uL)
- GG no insert control (5 uL)
- GG m1 (5 uL)
- *heat shock for 30 s instead of 45
- Recover at 37 C in 200 uL SOC
- *Didn’t have enough Cam plates so we used different concentrations
Sam
- Human Practices
Sara
- Cleaned out the fridge/bleached plates
- Worked on the lab notebook
- Overnight cultures of the Cas9 glycerol stocks
- Ran a miniprep for each tube of culture, one with water and one with elution buffer
- 89 ng/uL with water elution, 25 ng/uL with elution buffer elution
Shu
- Transformation with Paul
- Read papers
Tyler
- PCR to linearize mRFP:
- 2 x 50µL reactions:
- 1µL DMSO
- 1 µL mRFP backbone
- 1 µL Tet Lnrz for Cas9 FWD (10µM)
- 1 µL mRFP Reverse (10µM)
- 21 µL water
- Negative Control:
- 22 µL water
- 1µL DMSO
- 1 µL Tet Lnrz for Cas9 FWD (10µM)
- 1 µL mRFP Reverse (10µM)
- Conditions:
- Ran a gel on the linearized mRFP:
- Gibson Assembly of mRFP:
- 50 ng (0.40 µL) of mRFP backbone
- 16.6 ng (1.66 µL) of template gRNA gblock
- 7.94 µL of water
- 10µL of master mix
- Cleaned out fridge/bleached old plates
- Finished methicillin resistant gRNAs
- DpnI digest
- Run gel and gel extract
Paul
- PCR linearize Tet BB for GFP
- 3 tubes A: (In this order in the gel)
- Original template
- New template
- Half primer concentration (0.25 uL each 10 uM primer)
- 1 tube B: original template
- DpnI digest at 37 C for 2 hours
- Retransform:
- Gibson + control (5 uL)
- Gibson - control (no insert) (3 uL)
- pUC19 (1 uL, 50 pg/uL)
- GG no ligase control (5 uL)
- GG no insert control (5 uL)
- GG m1 (5 uL)
- *heat shock for 30 s instead of 45
- Recover at 37 C in 200 uL SOC
- *Didn’t have enough Cam plates so we used different concentrations
Sam
- Human Practices
Sara
- Cleaned out the fridge/bleached plates
- Worked on the lab notebook
- Overnight cultures of the Cas9 glycerol stocks
- Ran a miniprep for each tube of culture, one with water and one with elution buffer
- 89 ng/uL with water elution, 25 ng/uL with elution buffer elution
Shu
- Transformation with Paul
- Read papers
Tyler
- PCR to linearize mRFP:
- 2 x 50µL reactions:
- 1µL DMSO
- 1 µL mRFP backbone
- 1 µL Tet Lnrz for Cas9 FWD (10µM)
- 1 µL mRFP Reverse (10µM)
- 21 µL water
- Negative Control:
- 22 µL water
- 1µL DMSO
- 1 µL Tet Lnrz for Cas9 FWD (10µM)
- 1 µL mRFP Reverse (10µM)
- Conditions:
- 2 x 50µL reactions:
- Ran a gel on the linearized mRFP:
- Gibson Assembly of mRFP:
- 50 ng (0.40 µL) of mRFP backbone
- 16.6 ng (1.66 µL) of template gRNA gblock
- 7.94 µL of water
- 10µL of master mix
- Cleaned out fridge/bleached old plates
- Finished methicillin resistant gRNAs
