Team:Northwestern/09 15

Notebook

Thursday, September 15th

Tasks:

Michelle

  • Streaked JC8031 from glycerol stocks on Amp, Kan, Cam, Tet plates
  • Helped Tyler pick Golden Gates for AmiA
  • Plated cells for Cas9-gRNA cotransformation

Sam

  • Printed Auburn Gresham sheets
  • Updated OneNote
  • To do: design Auburn Gresham survey

Tasfia

  • Website content
  • Restriction Digest for Cas9 into pSB1C3 for part submission
    • Cas9 restriction digest
      • 4 μL Cas9 with added restriction sites (named “Cas9-Res-Dig,” 248.2 ng/μL)
      • 1 μL EcoRI-HF
      • 1 μL SpeI GQ
      • 5 μL 10X Cutsmart buffer
      • 39 μL nuclease-free water
      • Run for 60 minutes at 37°C, heat inactivated enzymes at 80°C for 20 minutes
    • pSB1C3 restriction digest
      • 4 μL linearized pSB1C3 (25 ng/μL)
      • 4 μL “Master Mix” as instructed by iGEM protocol
      • 2.5 μL Cutsmart buffer (looking back, should have used NEB Buffer 2)
      • 0.25 μL EcoRI-HF
      • 0.25 μL SpeI GQ
      • 9 μL nuclease-free water
      • 0.25 μL DpnI
      • Run for 30 minutes at 37°C, heat inactivated enzymes at 80°C for 20 minutes
  • Ligated Cas9 with pSB1C3 (two reactions to see which protocol works, if any)
    • iGEM ligation protocol
      • 2 μL pSB1C3 after restriction digest
      • 4.1 μL Cas9 digest (2:1 insert-to-backbone molar ratio)
      • 1 μL T4 ligase buffer
      • 0.5 μL T4 ligase
      • 2.4 μL nuclease-free water
      • Incubated at 16°C for 30 minutes, heat inactivated at 80°C for 5 minutes
    • Tyo lab ligation protocol
      • 4 μL pSB1C3 after restriction digest
      • 8.2 μL Cas9 digest (2:1 insert-to-backbone molar ratio)
      • 2 μL T4 ligase buffer
      • 1 μL T4 ligase
      • 4.8 μL nuclease-free water
      • Incubated at room temperature for 20 minutes, no heat inactivation (immediate transformation)
  • Transformed ligated products
    • 2 μL iGEM protocol ligated product
    • 2 μL Tyo protocol ligated product
    • 1 μL positive transformation control

Tyler

  • Picked colonies from GG plates
  • Talked to Jarod/picked up maxipreps that can be used for minipreps

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