Team:Northwestern/08 09

Notebook

Tuesday, August 9th

Tasks:

Paul

  • Plate with Patrick’s Cam cells grew ~50 colonies, only in the first couple streaks
  • Ran Gel salt test: Added X uL of 50 mM NaCl
    • Lane 1-4: Ladder: 4 uL, 2 uL, 1 uL, 4 uL diH2O; our blue dye
    • Lanes 5-7: Ladder: 4 uL, 2 uL, 1 uL; Jewett’s “blue” dye
    • Lanes 8-10: GFP 4: 4 uL, 2 uL, 1 uL,
  • Plated Patrick’s cells on different antibiotic plates
    • Prepared 30 ug/mL, 25 ug/mL, 20 ug/mL, and 12.5 ug/mL concentrations of Cam

Sara

  • Miniprepped the Cas9 Gibson products grown up on the 3 plates
    Tube Concentration (ng/uL) 260/280 260/230
    Gibson Cas9 1:3 136 1.73 1.05
    Gibson Cas9 1:3 (Shu) A 29 1.96 2.30
    Gibson Cas9 1:3 (Shu) B 222 1.89 2.38
    Gibson Cas9 1:2 (Shu) A 52 1.93 2.26
    Gibson Cas9 1:2 (Shu) B 152 1.91 2.30
  • Added plate pics to the lab notebook and got caught up on other lab notebook stuff
  • Helped Paul prepare plates to test cam concentrations

Shu

  • PCR mRFP (see Tyler’s note)
  • Made 4 small bottles of LB for plate test—100mL LB 1.5 Agar

Tyler

  • Submitted PO for Gibson Mix
  • Kept running BLAST of gRNA for MecA resistance, found more papers on KanR
  • PCR of the mRFP Backbone:
    • 2, 50µL reactions
      • 20µL water
      • 1 µL DMSO
      • 2µL mRFP backbone
      • 1µL Tet_Linear_for Cas9 Forward Primer
      • 1µL if mRFP reverse primer
      • 25µL of 2x Taq Master Mix
    • Negative Control (water)
      • 22µL water
      • 1 µL DMSO
      • 1µL Tet_Linear_for Cas9 Forward Primer
      • 1µL if mRFP reverse primer
      • 25µL of 2x Taq Master Mix

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