Team:Northwestern/08 22

Notebook

Monday, August 22nd

Tasks:

Jordan

  • Read vesicle entry paper
  • Transformed more sfGFP/MCherry minipreps from iGEM parts
    • Transformed 1 uL of each with 1 ul water as negative control
    • Heatshocked for 35 seconds
    • 200 uL of SOC
    • Plated 250 uL Tons of colonies
  • Looked into Periplasm fractioning tests and ethanol precipitation

Shu

  • Gel extracted Cas9-Lrz-SS (see gel in Tyler’s notes) Results: Everything had awful 260/230 ratios.

Tasfia

  • Re-ran PCR for GFP1 for GG, except with extended denaturation time
    • Two 50-µL reactions with different amounts of template
    • Template sfGFP concentration, when diluted 1:9 template-to-water, was 2.6 ng/µL
    • Reaction with 2.6 ng sfGFP
      • 25 µL OneTaq 2X Master Mix
      • 1 µL DMSO
      • 1 µL 10 uM forward primer for GFP
      • 1 µL 10 uM reverse primer for GFP
      • 1 µL sfGFP template
      • 21 µL nuclease-free water
    • Reaction with 1.3 ng sfGFP
      • 25 µL OneTaq 2X Master Mix
      • 1 µL DMSO
      • 1 µL 10 uM forward primer for GFP
      • 1 µL 10 uM reverse primer for GFP
      • 0.5 µL sfGFP template
      • 21.5 µL nuclease-free water
    • Conditions: 95°C (5:00) | 95°C (0:07), 54°C (0:10), 72°C (0:43) | 72°C (5:00)
  • Gel of the GFP1 PCR product
    • 20 µL PCR product in each well
    • Used purple loading dye and purple 2-log ladder at 1:3 ladder-to-dye ratio
    • There are still bands at ~400, where they usually occur, but we seem to have some bands near 1000 bp
    • Our gels consistently end up with products that are not where our ladder predicted them to be. From this we can conclude that the amount of template added to the PCR is not causing the bands to appear smaller than they actually are
  • Bands have been excised for extraction tomorrow:
    • stored in -20°C freezer
    • Put all the 1000-bp bands in two separate tubes
    • Also excised the ~400-bp bands because I was thinking about extracting that DNA as a gel extraction troubleshooting experiment
    • Masses are small because of 3D cutting
    • Masses:
      • Reaction with 2.6 ng, ~1000 bp: 0.0706g
      • Reaction with 1.3 ng, ~1000 bp: 0.0862g
      • Reaction with 2.6 ng, ~400 bp: 0.1295g
      • Reaction with 1.3 ng, ~400 bp: 0.1486g
  • Sent e-mails to:
    • Dr. Sutton from NU Medicine
    • Dr. Postelnick from NU Medicine
    • Emma Nechamkin from Auburn Gresham High School

Tyler

  • Ran PCR of Lnrzed Cas9
    • 2 x 50µL reactions:
      • 0.5 µL Template (conc ~ 2.5 ng/µL)/1µL Template
      • 1µL SS FWD (10µM)
      • 1 µL SS REV (10µM)
      • 1µL DMSO
      • 21.5µL water/ 22µL water
      • 25 µL Taq Master Mix
    • DpnI digest for overnight at room temperature
  • Ran gel of PCR product Linearized Cas9 for SS at different amounts of template
  • Read about carbenicillin resistance in E. Coli, new omv uptake paper, looked more into the pathways of antibiotic resistance in pathogens

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