Figure 1: Our SOC + positive transformation control

Figure 2: Kelly’s SOC + positive transformation control

Figure 3: Positive Gibson control

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