Team:Northwestern/09 08

Notebook

Thursday, September 8th

Western Blot Results:

Tasks:

Jordan

  • Helped with Western blot in the morning
  • Looked into alternative periplasm fraction options
  • Transformed Golden Gates and Gibsons
    • 1 uL of Patrick’s transformation control on Amp
    • 1 uL of Gibson YcdO product on Cam
    • 2 uL “ “ “
    • 3 uL “ “ “
    • 4 uL “ “ “
    • 3 ul of positive Golden Gate control from Patrick on Amp
    • 3 ul GG control w/ no ligase on Amp
    • 3 uL GG control w/ no insert on Amp
    • 3 uL gRNA-guide GG on Tet
    • 3 uL GFP-mcherry Golden Gate on Cam
    • Used 950 uL of SOC and plated 2 plates of each
    • Incubated for 1 hr. 15 minutes

Michelle

  • Ran gel on Cas9-SS Gibsons (2)
    • Completely empty
  • Made 40 fmol/uL dilutions of gRNA template, mCherry1, and Tet lnrz for GFP for GG Golden Gate assembly
  • Ran Golden Gate reactions
    • mCherry:
      • 1 uL of 40 fmol/uL mCherry1 PCR product
      • 1 uL of 40 fmol/uL Tet lrnz for GFP PCR product
      • 1 uL of 40 fmol/uL TorA
      • 2 uL of 10X T4 ligase buffer
      • 2.24 uL of 110uL BSA + 10uL T4 ligase buffer
      • 0.5 uL of 400,000U/mL T4 ligase from NEB
      • 0.5 uL of BsaI
      • Water to 21.76 uL
    • gRNA:
      • 1 uL of 40 fmol/uL gRNA template
      • 1 uL of annealed cutting guide insert from primers (straight from tube: ~ 6 uM)
      • 2 uL of 10X T4 ligase buffer
      • 2.24 uL of 110uL BSA + 10uL T4 ligase buffer
      • 0.5 uL of 400,000U/mL T4 ligase from NEB
      • 0.5 uL of BsaI
      • Water to 21.76 uL
    • Positive control:
      • 1 uL of Patrick’s positive control “32, 50ng”
      • 1 uL of Patrick’s positive control
      • 2 uL of 10X T4 ligase buffer
      • 2.24 uL of 110uL BSA + 10uL T4 ligase buffer
      • 0.5 uL of 400,000U/mL T4 ligase from NEB
      • 0.5 uL of BsaI
      • Water to 21.76 uL
    • Negative control (no ligase):
      • 1 uL of Patrick’s positive control “32, 50ng”
      • 1 uL of Patrick’s positive control
      • 2 uL of 10X T4 ligase buffer
      • 2.24 uL of 110uL BSA + 10uL T4 ligase buffer
      • 0.5 uL of BsaI
      • Water to 21.76 uL
    • Negative control (no insert):
      • 1 uL of Patrick’s positive control “32, 50ng”
      • 2 uL of 10X T4 ligase buffer
      • 2.24 uL of 110uL BSA + 10uL T4 ligase buffer
      • 0.5 uL of 400,000U/mL T4 ligase from NEB
      • 0.5 uL of BsaI
      • Water to 21.76 uL
    • Conditions:
      • 30 cycles, 37°C (2:00) 16°C (2:00) 50°C (5:00) 80°C (5:00)
  • In the future add 2 uL of 10X BSA in every reaction tube

Paul

  • Finished Western Blot procedure with Ben
  • TetR plasmid expressed less Cas9 than Top10
    • Fraction procedure may need revision:
      • Slight Cas9 residue in periplasm fraction
    • Lanes:
      • 1: ladder
      • 2: Whole cell ClyA-GFP
      • 3: Whole cell Neg control
      • 4: Whole cell Cas9 in pSB1C3 (Tet promoter)
      • 5: periplasm ClyA-GFP
      • 6: periplasm Neg control
      • 7: periplasm Cas9 in pSB1C3 (Tet promoter)
      • 8: ladder
  • Phosphorylate and anneal gRNA primers:
    • 1 uL PNK
    • 0.61 uL of 100 uM primer 1
    • 0.61 uL of 100 uM primer 2
    • 2 uL T4 ligase buffer
    • Water to 20 uL
    • @ 37°C for 30 min @ 95°C for 5 min
    • Cool to 28 C at ~1°C/min

Sam

  • Reached out to museums
  • Put finishing touches on pamphlet
  • Began writing human practices section for website
  • Began writing collaboration section for website
  • Emailed Australia

Sara

  • Read through the Australians’ protocols and made a list of questions and things that we would need to do them
  • Did a little lab notebook work
  • Plated the gibson Ycdo and Cas9 gibson products
    • 500 uL of the 1000 uL total solutions onto 2 plates each

Tyler

  • Mathematical modeling, looked into and researched other antibiotic resistant targets
  • Gibson Reaction (Ycdo)
    • 2.78 n.f. H20
    • 0.22 µL Ycdo Insert
    • 2 µL (36 ng) SS Lnrz Cas9
    • 5 µL Gibson Mix

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