Team:Northwestern/07 25

Notebook

Monday, July 25th

Agenda:

  • Check the GG storage vector
  • Freezer inventory
  • Figure out sequencing of Cas9
  • Review constructs
  • Human practices
  • Find protocols for next steps
  • Design sequencing primers for the GFP construct
  • Ligate GFP and mCherry parts into the storage vector
  • Look into Weinberg and Provost travel grants

Tasks:

Jordan

  • Discovered pSB1A3 contains BsaI site, not suitable for GFP storage vector
  • Looked for periplasmic lysis protocols
  • Scheduled call with Dr. Persell

Michelle

  • Freezer inventory & organization

Paul

  • Figured out sequencing submission process
  • Planned ligation
  • Planned gRNA plasmid

Sam

  • Emailed profs regarding Eligo Bioscience
  • Prepped questions for Dr. Persell
  • Website work
  • Troubleshot gel imager
  • Proposed that gels should be ran at a lower voltage for longer duration in order to prevent “band smiling”

Sara

  • PCR of GFP and mCherry
    • Miniprep of overnight BB part cultures:
      • GFP concentration: 39.7 ng/uL, 260/280: 2.02, 260/230: 1.44
      • mCherry concentration: 38 ng/uL, 260/280: 1.9, 260/230: 1.01
    • Procedure:
      • 2.5 uL 10X PCR buffer
      • 0.5 uL 10 mM DNTPs
      • 0.5 uL of forward primer at 10 mM
      • 0.5 uL of reverse primer at 10 mM
      • 0.5 uL template (Shu's miniprep GFP and mCherry products)
      • 0.25 Taq polymerase
      • 20.25 uL H20
    • PCR settings:
      • Denature: 95 C, 7 s.
      • Anneal: 51 C, 43 s.
      • Elongate 72 C, 2 m.
  • Made 400 mL 1% agarose with Shu
  • Poured 2 gels
  • Ran a gel of the PCR products
    • GFP3 and 4 did not have bands in the correct place, so we reran PCR on them and on GFP 2, which had a lower concentration and 260/230 ratio than the rest
    • We plan to troubleshoot by changing the voltage we’re running at and using newly diluted TAE

Shu

  • PCR of GFP and mCherry
    • Miniprep of overnight BB part cultures:
      • GFP concentration: 39.7 ng/uL, 260/280: 2.02, 260/230: 1.44
      • mCherry concentration: 38 ng/uL, 260/280: 1.9, 260/230: 1.01
    • Procedure:
      • 2.5 uL 10X PCR buffer
      • 0.5 uL 10 mM DNTPs
      • 0.5 uL of forward primer at 10 mM
      • 0.5 uL of reverse primer at 10 mM
      • 0.5 uL template (Shu's miniprep GFP and mCherry products)
      • 0.25 Taq polymerase
      • 20.25 uL H20
    • PCR settings:
      • Denature: 95 C, 7 s.
      • Anneal: 51 C, 43 s.
      • Elongate 72 C, 2 m.
  • Made 400 mL 1% agarose with Sara
  • Poured 2 gels
  • Ran a gel of the PCR products
    • GFP3 and 4 did not have bands in the correct place, so we reran PCR on them and on GFP 2, which had a lower concentration and 260/230 ratio than the rest
    • We plan to troubleshoot by changing the voltage we’re running at and using newly diluted TAE

Tasfia

  • PCR on miniprepped INP
  • Ran gel on INP PCR product
  • Web content

Tyler

  • Designed gRNA template w/homology to mRFP plasmid, mRFP insert with correct GG sticky ends, reverse primer
  • Sponsors comm (Bio-rad)

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