Team:Northwestern/07 26

Notebook

Tuesday, July 26th

Agenda:

  • Troubleshoot uneven gel running
  • Review constructs
  • Human practices
  • Find protocols for next steps
  • Ligate GFP and mCherry parts into the storage vector
  • Look into Weinberg and Provost travel grants

Tasks:

Jordan

  • Reran sfGFP and mCherry PCR with Sara
    • 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
  • Funds organization
  • Reworked CLP letter

Michelle

  • Made Kan Plates:
    • 350 mL LB
    • 1.75 mL Kanamycin (10 mg/mL) (to 50 ug/mL working concentration)
    • Poured 1.25 sleeve’s worth of plates
  • Made Tet Plates
    • 300 mL LB
    • 0.6 mL Tetracycline (5 mg/mL) (to 10 ug/mL working concentration)
    • Poured 1 sleeve’s worth of plates
  • Made LB Stock
    • 10.03 g Tryptone
    • 1 mL 1N NaOH
    • 5.02 g Bacto Yeast extract
    • 5.04 g NaCl
  • Made 1X TAE
    • 100 mL 10X TAE
    • 900 mL dH20

Paul

  • Sent Cas9 minipreps to sequencing
  • Ran gels on GG PCR products (sfGFP & mCherry)
  • Ran PCR on GFP&mCherry (Ta=56¯C)
  • Sequencing Premix Protocol (use small 600 uL tubes)
    • 1 template+1 primer per tube
      • 450-600 ng of DNA template (~100-800)
      • 1.2 uL of 10 uM primer
      • Water to 15 uL
  • Submit sequencing through Genesifter account

Sara

  • Reran sfGFP and mCherry PCR with Sara
    • 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
  • Loaded the samples into the gels with Paul
  • Troubleshot too-concentrated primers in PCR reaction

Shu

  • Transformation of mRFP in pSB1T3
  • Sent Tyler Josh’s slides

Tasfia

  • Cleaned up miniprepped INP PCR product (80.2 ng/uL final concentration)
  • Autoclaved LB
  • Poured Tet plates (see Michelle’s notes for details)

Tyler

  • Sequencing Cas9 preparation
  • Reviewed SS Primers, created more mRFP guides, sent to Kelly
  • Began reviewing Josh’s modeling slides from Shu, emailed Joe about meeting time

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