Team:Northwestern/08 24

Notebook

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

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