Tuesday, August 9th
Tasks:
Paul
- Plate with Patrick’s Cam cells grew ~50 colonies, only in the first couple streaks
- Ran Gel salt test: Added X uL of 50 mM NaCl
- Lane 1-4: Ladder: 4 uL, 2 uL, 1 uL, 4 uL diH2O; our blue dye
- Lanes 5-7: Ladder: 4 uL, 2 uL, 1 uL; Jewett’s “blue” dye
- Lanes 8-10: GFP 4: 4 uL, 2 uL, 1 uL,
- Plated Patrick’s cells on different antibiotic plates
- Prepared 30 ug/mL, 25 ug/mL, 20 ug/mL, and 12.5 ug/mL concentrations of Cam
Sara
- Miniprepped the Cas9 Gibson products grown up on the 3 plates
Tube
Concentration (ng/uL)
260/280
260/230
Gibson Cas9 1:3
136
1.73
1.05
Gibson Cas9 1:3 (Shu) A
29
1.96
2.30
Gibson Cas9 1:3 (Shu) B
222
1.89
2.38
Gibson Cas9 1:2 (Shu) A
52
1.93
2.26
Gibson Cas9 1:2 (Shu) B
152
1.91
2.30
- Added plate pics to the lab notebook and got caught up on other lab notebook stuff
- Helped Paul prepare plates to test cam concentrations
Shu
- PCR mRFP (see Tyler’s note)
- Made 4 small bottles of LB for plate test—100mL LB 1.5 Agar
Tyler
- Submitted PO for Gibson Mix
- Kept running BLAST of gRNA for MecA resistance, found more papers on KanR
- PCR of the mRFP Backbone:
- 2, 50µL reactions
- 20µL water
- 1 µL DMSO
- 2µL mRFP backbone
- 1µL Tet_Linear_for Cas9 Forward Primer
- 1µL if mRFP reverse primer
- 25µL of 2x Taq Master Mix
- Negative Control (water)
- 22µL water
- 1 µL DMSO
- 1µL Tet_Linear_for Cas9 Forward Primer
- 1µL if mRFP reverse primer
- 25µL of 2x Taq Master Mix
Paul
- Plate with Patrick’s Cam cells grew ~50 colonies, only in the first couple streaks
- Ran Gel salt test: Added X uL of 50 mM NaCl
- Lane 1-4: Ladder: 4 uL, 2 uL, 1 uL, 4 uL diH2O; our blue dye
- Lanes 5-7: Ladder: 4 uL, 2 uL, 1 uL; Jewett’s “blue” dye
- Lanes 8-10: GFP 4: 4 uL, 2 uL, 1 uL,
- Plated Patrick’s cells on different antibiotic plates
- Prepared 30 ug/mL, 25 ug/mL, 20 ug/mL, and 12.5 ug/mL concentrations of Cam
Sara
- Miniprepped the Cas9 Gibson products grown up on the 3 plates
Tube Concentration (ng/uL) 260/280 260/230 Gibson Cas9 1:3 136 1.73 1.05 Gibson Cas9 1:3 (Shu) A 29 1.96 2.30 Gibson Cas9 1:3 (Shu) B 222 1.89 2.38 Gibson Cas9 1:2 (Shu) A 52 1.93 2.26 Gibson Cas9 1:2 (Shu) B 152 1.91 2.30 - Added plate pics to the lab notebook and got caught up on other lab notebook stuff
- Helped Paul prepare plates to test cam concentrations
Shu
- PCR mRFP (see Tyler’s note)
- Made 4 small bottles of LB for plate test—100mL LB 1.5 Agar
Tyler
- Submitted PO for Gibson Mix
- Kept running BLAST of gRNA for MecA resistance, found more papers on KanR
- PCR of the mRFP Backbone:
- 2, 50µL reactions
- 20µL water
- 1 µL DMSO
- 2µL mRFP backbone
- 1µL Tet_Linear_for Cas9 Forward Primer
- 1µL if mRFP reverse primer
- 25µL of 2x Taq Master Mix
- Negative Control (water)
- 22µL water
- 1 µL DMSO
- 1µL Tet_Linear_for Cas9 Forward Primer
- 1µL if mRFP reverse primer
- 25µL of 2x Taq Master Mix
