Wednesday, August 24th
Transformation Results:
Gibson gRNA plate had no colonies, but all the positive controls (shown below) worked.
Figure 1: Our SOC + positive transformation control
Figure 2: Kelly’s SOC + positive transformation control
Figure 3: Positive Gibson control
Tasks:
Jordan
- Plated cytoplasmic GFP, TetR-Cas9, and positive control transformations
- TetR-Cas9 on big plate, plated 250 uL
- Other two on small plates with 100 uL
- Made some 3M sodium acetate solution to try ethanol precipitation—Kelly says it’s worth a try—see Paul for final pH
- Induced ClyA culture
- 500 uL of 20% Arabinose (.2% final) at about 5:15 p.m. (culture stop incubating at ~3:45, was on ice for 1.5 hours)
- Incubating at RT overnight
- Made and autoclaved buffers and NaOAc from yesterday
- Cell fractioning buffer 1: 200 mL of .2 M Tris-HCl (pH 8) (40 mL), 200 g/L sucrose (40g), .1 M EDTA (5.84 g), water up to 200 mL
- Soft Cell fraction buffer 2: .01 M Tris HCl (pH 8) (1 mL), .005 M MgSO4 (.06 g), water up to 100 mL
- Harsh cell fraction buffer 2: previous plus .2 % SDS (.2 g), 1% Triton X-100 (100 uL), water up to 100 mL
- L-arabinose: 20% w/v (100X) stock solution, 1g in 5 mL
Paul
- Grew ClyA-GFP fusion/DeLisa control
- Control OD=0.9
- GFP OD=.45
- Transfect Kelly’s TetR cells with our saCas9
- Transformed:
- Interlab Test 1 so we have cytosolic GFP reporter (100pg)
- Positive control (Cell Efficiency test 50 pg)
- *Into Kelly’s TetR cells*: Assembled Cas9 (~42 ng)
- Planned Buffers that Jordan made (see Jordan’s notes)
- Planned Western Blot procedure
- Two 5 mL cultures of our cells (Top10-constitutive)-glycerol stocks
- Grow to OD=0.5-0.6
- Two 5 mL cultures of Kelly’s cells (TetR-inducible)-Transformed today
- Grow to OD=0.5-0.6
- Induce with tet inducer
- Express overnight at 18°C
- Grow up over Sunday night (glycerol stock swab/pick colonies)
- Bring 5 mL cultures to Ben in Jewett lab to start Western Blot procedure
Tasfia
- Gel salt ladder test
- *NaCl solution dilutions made with diH2O and the already-prepared 50mM NaCl solution from 9 August 2016
- **Loading dye used: Jewett lab blue 6X dye
- Moral of the story: our ladders are inaccurate and there’s hope in our PCRs
- Kelly gave us [some unspecified large amount] of Leonard lab ladder, so we should be set for awhile
- Kelly’s 3 kb sample reads at 3 kb on her ladder
- Cas9-Lrz-SS reads at 5-6 kb on the Leonard ladder
- GFP1 reads at 700-800 bp on Leonard ladder so it might be correct
Tyler
- Inoculated 50 mL cultures
- Pictures of the results of the previous day transformation
- Designed/ordered primers to amplify G-blocks
