Team:Northwestern/08 05

Notebook

Friday, August 5th

Tasks:

Michelle

  • Made 50 μL aliquots of nfH2O
  • Made more femtomole dilutions
    • Tet backbone: 65.1 ng/μL stock = 50.17 fmol
      • 7.97 μL of stock to 10 μL
    • mCherry 1: 65.5 ng/μL stock = 132.5 fmol
      • 3.02 μL stock to 10 μL
    • TorA: 100μM stock
      • First 1:100 dilution (done to 50 μL)
      • Second 1:25 dilution (done to 50 μL)
  • Transformed reactions
    • Transformed 5 μL of DNA solution to 50 μL of cells for all transformations
    • Recovered in 200 μL of SOC media
    • Transformation controls:
      • NEB cells: mRFP in pSB1C3 (Cam plate)
      • Our cells:
        • mRFP in pSB1C3 (Cam plate)
        • mRFP in pSB1T3 (not plated because we ran out of Tet plates)
    • Golden Gate reactions:
      • NEB cells: mCherry 1 + TorA (Cam plate)
      • Our cells:
        • mCherry 1 + TorA (Cam plate)
        • mCherry 1 + TorA without ligase (Cam plate)
        • Just linearized Tet backbone, no insert (Cam plate)
    • Gibson reactions:
      • NEB cells: Gibson for Cas9 (Cam plate)
      • Our cells:
        • Gibson for Cas9 (Cam plate)
        • Gibson kit positive control (Amp plate)
    • Golden Gate storage vector ligation reaction:
      • NEB cells: mCherry 1 (Tet plate)
      • Our cells:
      • mCherry 1 (not plated because we ran out of Tet plates)
      • mCherry 2 (Tet plate)
      • mCherry 3 (Tet plate)
      • mCherry 4 (Tet plate)
      • mCherry 5 (Tet plate)
      • GFP 1 (Tet plate)
      • GFP 2 (Tet plate)
      • GFP 3 (Tet plate)
      • GFP 4 (Tet plate)

Sara

  • Made cam plates
    • 300 mL LB
    • 4.5 g bacto agar
    • Autoclave, then 300 uL Cam

Shu

  • Gibson assembly with Tyler
    • Cas9 Part
      • 5.8µL of water
      • 2.4µL backbone
      • 0.93 µL Cas9-1
      • 0.86µL Cas9-2
      • 10µL of master mix
    • Positive Control
      • 10µL of positive control
      • 10µL of master mix

Tasfia

  • Ran restriction digest of Gibson Assembly Cas9 products with XbaI (25-μL reactions)
    • “Cas9” (conc’n: 1370 ng/μL; 260/280: 2.33; 260/230: 1.79)
      • 0.75 μL DNA
      • 2.5 μL Cutsmart 10x buffer (#B7204S exp 6/17)
      • 1 μL XbaI
      • 20.75 μL nf water
    • 1:3 backbone-to-insert product (conc’n: 1102 ng/μL; 260/280: 2.34; 260/230: 1.83)
      • 0.93 μL DNA
      • 2.5 μL Cutsmart 10x buffer
      • 1 μL XbaI
      • 20.57 μL nf water
    • 1:2 backbone-to-insert product (conc’n: 1080 ng/μL; 260/280: 2.33; 260/230: 1.78)
      • 0.95 μL DNA
      • 2.5 μL Cutsmart 10x buffer
      • 1 μL XbaI
      • 20.55 μL nf water
      • Incubated digest for ~45 minutes at 37°C
    • Results:
      Gibson Product Concentration (ng/uL) 260/280 260/230
      Cas9 50.2 2.10 0.77
      1:3 47.8 1.95 0.54
      1:2 47.5 1.91 0.51
  • Ran a gel on digest products, Gibson inserts, and some golden gate products (Thush)
    • (mCherry1 (tat?), mCherry2 (tat? first row on the GG purple tube holder), mCherry1 control
    • 6X blue loading dye (NEB #B7021S, exp 6/14) was used
      • Wells 1 and 10: Two ladders ~ 1:3 2-log purple ladder-to-loading dye dilution (8 μL per well)
      • Well 2: 1:2 backbone-to-insert Gibson product (6 μL)
      • Well 3: 1:3 backbone-to-insert Gibson product (6 μL)
      • Well 4: “Cas9” Gibson product (6 μL)
      • Well 5: Cas9(1) insert (9.6 μL)
      • Well 6: Cas9(2) insert (10.8 μL)
      • Well 7: mCherry1 tat (6 μL)
      • Well 8: mCherry2 tat (6 μL)
      • Well 9: mCherry1 control (6 μL)
    • Ran at 95 V for 48 minutes
  • Gel looked strange; Gibson products appeared nowhere on the gel, even though the NanoDrop picked up good concentrations.
  • Troubleshooting
    • We let the gel harden too long
    • Our ladder is just bad
    • Cas9(1) and Cas9(2) show up at ~3kb, when they should be at 1.8 and 1.6, respectively
    • Gibson product has a lot of contaminants (low 260/230 ratios on all three samples)
    • Diagonal line for “lighter” mCherry bands due to unequal distribution of electric field
    • GG products were also not linearized
    • Didn’t NanoDrop that before loading gel either

Tyler

  • Gibson assembly with Shu
  • Helped with Michelle's transformations

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