Tuesday, August 16th
Results:
The only transformation that was successful was the positive transformation control from the NEB competent cells box.
Tasks:
Jordan
- Loaded linearized Cas9 gel
- Ran linearized for SS cas9 backbone gel
- Ran gel at 95 V for ~1 hr 20
- Transformed Gibson reactions
- 3 ul of each, 3 ul of 50 pg/ul Competent Cell test RFP, 3 ul nf water
- Thawed cells 15 minutes before transformation
- On ice for slightly more than 30 minutes
- Heat shocked by Sam
- Added 200 uL of SOC (maybe slightly less chilled because working near flame?)
- Incubated 1 hour 15 minutes
- Plated 250 uL
Michelle
- Made glycerol stocks of Cas9 grown w/ antibiotic (colony A) and Cas9 grown without antibiotic
- 500 uL of 50% glycerol
- 500 uL of cell culture
- Miniprepped Cas9 cultures and Tet backbone cultures
- Cas9 grown in antibiotic (colonies A, B, C)
- Cas9 grown without antibiotic
- Tet backbone (colonies A, B, C)
- Nanodrop results:
- Tet plasmid colony A: 450.9 ng/uL, 260/280: 1.88, 260/230: 2.52
- Tet plasmid colony B: 107.4 ng/uL, 260/280: 1.76, 260/230: 1.16
- Tet plasmid colony C: 125.3 ng/uL, 260/280: 1.93, 260/230: 3.07
- Cas9 w/ antibiotic colony A: 147.1 ng/uL, 260/280: 1.63, 260/230: 0.78
- Cas9 w/ antibiotic colony B: 218.1 ng/uL, 260/280: 1.58, 260/230: 0.72
- Cas9 w/ antibiotic colony C: 101.0 ng/uL, 260/280: 1.66, 260/230: 0.80
- Cas9 w/o antibiotic: 42.4 ng/uL, 260/280: 1.83, 260/230: 1.40
- Made LB
- 10.0190 g tryptone peptone
- 5.0098 g yeast extract
- 5.0065 g NaCl
- 1 mL 1 N NaOH
- Used 100 mL for expression culture
- Used 200 mL for plates
- Added 3.0043 g Bacto agar
- Poured 200 mL of Cam plates (0.2 mL Cam 1000X added)
- Gel extracted Tet linearized for GFP
- Part 1 + 2: 0.2783 g (0.8349 mL GEX buffer added)
- Nanodrop results: 14.2 ng/uL, 260/280: 2.02, 260/230: 0.87
- Gel extracted Tet linearized for gRNA
- Part 1: 0.3604 g (1.0812 mL GEX buffer added)
- Part 2: 0.3837 g (1.1511 mL GEX buffer added)
- Nanodrop results: 125.7 ng/uL, 260/280: 2.02, 260/230: 1.94
Sam
- Nanodrop data
- Suspensions of gene blocks were 5 ng/uL instead of 10 and had awful ratios. 3 to 5 = 260/280, -0.4ish = 260/230.
- Kelly said to trust the nanodrop and run the Gibson assuming they’re actually 5 ng/uL
- Something was 5500 ng/uL. Ratios were good
- Solution: dilute with nfH2O
- Worked on the phone call script with Jordan
- No response from yesterday’s call. Called more numbers.
- Performed gel extraction of linearizing Cas9 gel.
- Accidentally forgot to let elution water sit for five minutes. Concentration is still fine. 260/230 is 0.8
Sara
- Updated the lab notebook and nagged people about writing down the things they’ve done
- Checked OD of the cas9 cultures we’re growing up
Shu
- Gibson Reactions
- Cas9-TorA (vector:insert=1:5)
- 0.64uL backbone
- 1.11uL TorA gblock
- 3.25uL water
- 5uL master mix Cas9-INP (vector:insert=1:3)
- 0.64uL backbone
- 0.65uL diluted INP
- 3.71uL water
- 5uL master mix mRFP backbone-gRNA
- 1.05uL diluted mRFP
- 1.66uL template cutting gRNA gblock
- 2.29uL water
- 5uL master mix
- Positive control
- 5uL positive control
- 5uL master mix
- Negative control
- 0.64uL backbone
- 1.11uL TorA gblock
- 8.25uL water
- Re-nanodrop linearized Cas9: concentration: 39ng/uL
Tyler
- PCR- Linearizing Cas9 for SS
- 2 x 50µL reactions:
- 21.5 µL water
- 1 µL DMSO
- 0.5 µL cas9 1:3
- 1 µL SS_Lnrz_Fwd
- 1 µL SS_Lnrz_Rev
- 25 µL Taq Master Mix
- Negative Control:
- 22 µL water
- 1 µL DMSO
- 1 µL SS_Lnrz_Fwd
- 1 µL SS_Lnrz_Rev
- 25 µL Taq Master Mix
- Conditions:
- DpnI digest for 2 hours at 37°C
- 2 x 50µL reactions:
- Ran Calculations for Gibson
- Began loading gel for PCR product, let Jordan finish
- Nanodropped some of our gblocks/troubleshot DNA concentrations
- Took a screwdriver to the ice in the fridge
