Team:Northwestern/08 15

Notebook

Monday, August 15th

Agenda:

  • Glycerol stocks of Cas9
  • Miniprep Cas9
  • Miniprep Tet BB
  • PCR Linearize Tet for GFP
  • PCR mRFP
  • Retransform Cas9-with-SS Gibson
  • PCR linearize for ClyA
  • Clean out fridge
  • PCR linearize mRFP backbone
    • DpnI digest
    • Run gel and gel extract
  • Gibson the gRNA with template
  • Transform

Tasks:

Paul

  • PCR linearize Tet BB for GFP
    • 3 tubes A: (In this order in the gel)
      • Original template
      • New template
      • Half primer concentration (0.25 uL each 10 uM primer)
    • 1 tube B: original template
    • DpnI digest at 37 C for 2 hours
  • Retransform:
    • Gibson + control (5 uL)
    • Gibson - control (no insert) (3 uL)
    • pUC19 (1 uL, 50 pg/uL)
    • GG no ligase control (5 uL)
    • GG no insert control (5 uL)
    • GG m1 (5 uL)
    • *heat shock for 30 s instead of 45
    • Recover at 37 C in 200 uL SOC
    • *Didn’t have enough Cam plates so we used different concentrations

Sam

  • Human Practices

Sara

  • Cleaned out the fridge/bleached plates
  • Worked on the lab notebook
  • Overnight cultures of the Cas9 glycerol stocks
    • Ran a miniprep for each tube of culture, one with water and one with elution buffer
    • 89 ng/uL with water elution, 25 ng/uL with elution buffer elution

Shu

  • Transformation with Paul
  • Read papers

Tyler

  • PCR to linearize mRFP:
    • 2 x 50µL reactions:
      • 1µL DMSO
      • 1 µL mRFP backbone
      • 1 µL Tet Lnrz for Cas9 FWD (10µM)
      • 1 µL mRFP Reverse (10µM)
      • 21 µL water
    • Negative Control:
      • 22 µL water
      • 1µL DMSO
      • 1 µL Tet Lnrz for Cas9 FWD (10µM)
      • 1 µL mRFP Reverse (10µM)
    • Conditions:
  • Ran a gel on the linearized mRFP:
  • Gibson Assembly of mRFP:
    • 50 ng (0.40 µL) of mRFP backbone
    • 16.6 ng (1.66 µL) of template gRNA gblock
    • 7.94 µL of water
    • 10µL of master mix
  • Cleaned out fridge/bleached old plates
  • Finished methicillin resistant gRNAs

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