Team:Northwestern/07 20

Notebook

Wednesday, July 20th

Agenda:

  • Meeting with Traci
  • Read the Golden Gate stuff from Patrick
  • Call millipore
  • Gibson of Cas9 parts and tet backbone
    • Transform the Gibson products
    • Miniprep
    • Sequencing
    • Linearize
  • Meet with Patrick
  • gRNA construct
  • Look into Weinberg and Provost travel grants
  • Fun antibiotic activity design

Tasks:

Jordan

  • Read GG protocol papers
  • Met with Traci from iGEM and UChicago

Sam

  • Continued compiling list of experts
  • Met with Prof. Broadbelt
  • Ran transformation (no controls):
    • Thawed 100 uL of comp cells
    • Added 5 uL of Gibson results to the comp cells
      • 15 uL of Gibson left over
      • Will run a gel with a little of it tomorrow
    • Mix sat 30 min on ice
    • 45 sec in water bath at 42°C
    • 5 min back on ice
    • 900 uL SOC broth added under flame
    • Shaking incubator at 37°C for 1 hr
    • Pipetted 50 uL and 100 uL onto 2 (total) Cam plates and let grow overnight
    • Plates were a little on the cold side when we plated
  • Gel:
    • 50 mL recycled gel
    • 5 uL SybrGreen
    • Did not cover while solidifying. Not used to covering because SyberSafe isn’t as sensitive

Sara

  • Patrick showed us how to phosphorylate the ends of the primer dimers/make them anneal to each other in prep for Golden Gate
  • Peter helped us run through the Gibson
  • Resuspended Cas9(1) and Cas9(2) in 10nL of nuclease free water, bring concentration down to 100 ng/uL
  • Gibson assembly

Shu

  • Resuspended Cas9 parts in water with Sara
    • Used 10 uL of the Gibson master mix
    • 6.25 uL NF water
    • 0.7 uL linearized Tet backbone
    • 1.6 uL Cas9(1)
    • 1.45 uL Cas9(2)
    • 1 hr at 50°C
    • Total of 20 uL
  • Learned Golden Gate with Patrick
  • Gibson assembly with Peter
  • Golden Gate paper reading

Tasfia

  • Gibson assembly (see Sara’s entry)
  • Started transformation (see Sam’s notes about transformation)
  • Looked into experts for human practices

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