Team:Northwestern/07 27

Notebook

Wednesday, July 27th

Agenda:

  • Pour 2 gels for GFP and mCherry
  • PCR linearization of the Tet-Cas9 backbone in preparation for insertion of ClyA or INP/signaling sequences
  • PCR Cleanup of GG gels

Tasks:

Jordan

  • Designed ClyA sequencing primers
  • Ran gel extraction/cleanup on mCherry’s (Shu did GFPs), followed kit procedure, used 30 ul water not elution buffer, added more buffer up to about 500 ul
  • Looked into more fractionation procedures Ligation

Michelle

  • Made glycerol stocks of A1–A3, A5, B1–B3, B5 Cas9 cultures
    • 500 μL overnight culture
    • 500 μL 50% glycerol in dH2O
    • In microcentrifuge tube stored in -80°C in Mordacq’s lab, in box marked 2016 iGEM Glycerol Stocks
  • Researched Cas9 expression & purification protocols
  • Cas9–INP and Cas9–ClyA Gibson math
  • Made 5mL cultures of Cas9 from glycerol stocks with Sara
    • 50 μL Cam in 5 mL LB
    • Cultures A3, A5, B2
  • Ran mCherry & sfGFP PCR
    • 0.5 μL of 10 μM forward primer
    • 0.5 μL of 10 μM reverse primer
    • 1 μL template (sfGFP or mCherry miniprep)
    • 12.5 μL of OneTaq Master Mix
    • 10.5 μL nuclease-free water
    • 95°C (2:00) | 95°C (0:07),  51°C (0:10),  72°C (0:43) | 72°C (5:00)
    • 25 μL reaction volume, 25 cycles
  • Aliquoted 100 mL of LB for autoclaving to grow up Cas9 tomorrow
  • Aliquoted LB into smaller bottles for autoclaving tomorrow

Paul

  • Looked up Cas9 expression procedure
  • Ran gel on mCherry and GFP PCR products
  • Helped out with various experiments

Sara

  • PCR of Tet-Cas9 backbone with Tyler
    • Used DNA from B2 and A5 overnight cultures
  • Made 5mL cultures of Cas9 from glycerol stocks with Michelle
    • 50 μL Cam in 5 mL LB
    • Cultures A3, A5, B2

Shu

  • Gel extracted sfGFP for Golden Gate assembly
  • Grew mRFP culture
  • Looked over SS sequencing primers

Tasfia

  • Made glycerol stocks with Michelle
  • PCR Cleanup of GFP/mCherry

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