Team:Northwestern/09 20

Notebook

Tuesday, September 20th

Tasks:

Jordan

  • Helped make competent Delisa cells
  • Ligated Cas9 and pSB1C3 restriction digests
    • 25 ng pSB1C3 (2ul)
    • 37 ng Cas9 insert (1.86 uL)
    • 1 ul T4 ligase buffer
    • 0.5 ul T4 ligase
    • 4.64 ul water
    • 1:1 insert:vector
    • 16°for 30 minutes, heat kill 80°C 20 minutes

Sam

  • Electroporated 4 lines of cells with Paul
  • Skipped the negative control because the Jewett lab only had 4 electroporation cuvettes

Sara

  • Poured a gel to screen the clyA-PET28a PCR
    • 3 uL sybersafe
    • 5 uL product, 1 uL dye
    • 6 uL ladder, 3 uL dye
    • The gel showed that the PCR did not work
  • Made more 1% agarose (400 mL)

Tasfia

  • Made competent cells out of DeLisa strain (with Paul and Jordan)
  • Transformed ligation product (Cas9 into pSB1C3 iGEM backbone for part submission), 3 μL DNA
  • Transformed pSB1C3 competent cell test plasmid to examine efficiency of DeLisa competent cells
  • Picked more colonies of gRNA+Cas9 cotransformation, and with existing overnight cultures made 2.5-mL aliquots to give cultures more air in the tubes

Tyler

  • Gibson Reactions
    • Cas9
      • 0.74 µL backbone (1:2 dilution)
      • 1.15 µL insert (1:5 dilution)
      • 3.11 µL water
      • 5 µL mix
    • SS-Cas9 DsbA
      • 0.74 µL backbone
      • 0.75 µL insert (1:5 dilution)
      • 3.51 µL water
      • 5 µL mix
    • SS-Cas9 Ycdo
      • 0.74 µL backbone
      • 0.95 µL insert (1:5 dilution)
      • 3.31 µL water
      • 5 µL mix Backbone
    • (-) control
      • 0.74 µL backbone
      • 4.26 µL water
      • 5µL mix

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