Team:Northwestern/08 25

Notebook

Thursday, August 25th

Tasks:

Jordan

  • Ran the cell fractions on the plate reader with Michelle
    • 485/520 fluorescence (rows = replicates)
    • No longer have linearized Cas9 for SS gel extract from 8.19 and 8.22 - lost the DNA in the ethanol precipitation
  • Ethanol precipitation of 8.19 and 8.22 linearized cas9 for SS gel extract
    • Added 1/10 the sample volume of 3M sodium acetate (2 uL for each 20 uL sample)
    • Added 2.5 volumes of 100% ethanol (added 50 uL of 95% ethanol (didn’t make a difference)) to each sample
    • Froze for 1 hr at -80°C
    • Centrifuge at max speed (14,000 rpm) for 30 minutes
    • Decant or pipette off supernatant
    • Air dry pellet for 15 minutes (then 30 in heat block)
    • Resuspend in water, vortex and spin down
    • Negative DNA was nanodropped
    • Lost the DNA

Michelle

  • Cleared unnecessary items from freezer
  • Made a 1000X dilution of Cam
    • 0.51 mL of 100 mg/mL stock diluted to 1.5 mL using 200-proof ethanol
  • Made more Cam plates
    • 200 mL LB + 3.0097 g Bacto Agar (autoclaved)
  • PCR signal sequences to add more homology
    • Nanodrop:
      • FhuD: 13.6 ng/uL
      • AmiA: 10.9 ng/uL
      • DsbA: 11.3 ng/uL
      • YcdO: 18.6 ng/uL
      • TorA: 6.5 ng/uL
      • NapA: 10.0 ng/uL
    • Mix:
      • 0.5 uL of template (SS)
      • 1.0 uL of fwd primer 10 uM
      • 1.0 uL of rev primer 10 uM
      • 1.0 uL of DMSO
      • 21.5 uL of nfH2O
      • 25 uL of OneTaq 2X Master Mix
  • Conditions:
  • Cut out gRNA and SS bands and froze them
  • 200 mL LB + 3.0097 g Bacto Agar (autoclaved)
  • Ran plate reader on periplasm fractions with Jordan

Paul

  • Tested cell fractioning procedure from Bielfeld iGEM 2014 with Tasfia
    • 2 x 20 mL of DeLisa line
    • 2 x 20 mL of ClyA-GFP in DeLisa line
    • Tested no SDS and SDS in Cell Frac. Buffer 2
    • *Final Supernatant contains periplasmic proteins
  • Cas9 Expression Protocol Plan:
    • Saturday:
      • Inoculate 4x 1 mL cultures for overnight growth (add appropriate antibiotic)
      • Inoculate 2 cultures of saCas9 with our glycerol stocks
      • Pick 2 colonies from plate in fridge (“TetR, Kelly’s cells) from 8-24-16
    • Sunday:
      • Inoculate/grow 5 mL cultures up to OD=0.5
      • Add 500 uL of grown up cultures to 5 mL LB with antibiotic
      • Grow to OD=0.5-0.6 (roughly)
      • Put on ice once at correct OD
      • Express overnight (~16 hrs) at 37 or 18°CC (1 culture of our cells and one culture of TetR Kelly’s cells at 18, and 1 culture of each cell line at 37)
      • Use Mordaq’s incubator for 18°C
    • Monday: Take 5 mL cultures to Ben in Jewett lab in morning (~9 am)

Shu

  • Ran gel on on Cas9-Lrz-SS:
    • Excised gel and froze for two hours, then extracted
    • Concentrations: see 8.29.16

Tasfia

  • Performed cell fractioning procedure with Paul (see his notes)
  • Re-ran PCR for Cas9 linearized for signal sequences (to DpnI digest and gel extract next morning)
    • 25 uL OneTaq 2X Master Mix
    • 1 uL DMSO
    • 1 uL 10 uM forward primer
    • 1 uL 10 uM reverse primer
    • 1 uL template
      • Concentration: 28 ng/uL
      • Amounts used:
        • 15 ng
        • 1.5 ng
    • 21 uL nuclease-free water

Tyler

  • Ran gRNA and Tet-Lrz-GFP PCRs

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