Team:Northwestern/09 09

Notebook

Friday, September 9th

Western Blot Results:

Tasks:

Jordan

  • Cleaned out the fridge, got rid of Interlab stuff, the base Cas9 plates that we have minipreps and glycerol stocks of, and controls and stuff that never worked anyway
  • Transformed 3 uL of DsbA Gibson from Gene blocks and from added homology PCRs, negative control with only backbone, and 1ul (50 pg) of competent cell test RFP
    • 30 second heat shock, 950 uL SOC, 1 hr 15 minute incubation, plated 2 plates of each

Paul

  • Added Golden Gate transformation pictures to slideshow
  • Presented at meeting with Profs

Sam

  • Emailed Dr. Tullman-Ercek
  • Made a gel
  • Updated OneNote
  • Transformation with Jordan

Sara

  • PCR to extend SS homology
    • 50 uL reaction of every SS we have
    • 0.5 uL template (SS)
    • 1.0 uL fwd primer @ 10 mM
    • 1.0 uL rev primer @ 10 mM
    • 1.0 uL DMSO
    • 21.5 uL dH20
    • 25 uL Onetaq 2X MM
  • PCR purified signal sequences

Tasfia

  • PCR: Linearization of Cas9 for signal sequences (again)
    • 50 uL reaction of every SS we have
    • 1 uL template (“Cas9 1:3 136 ng/uL” nanodropped 133 ng/uL)
      • 13 ng (“Cas9-Lrz-SS ‘A’”)
      • 1.3 ng (“Cas9-Lrz-SS ‘B’”)
    • 1 uL fwd 10 uM primer
    • 1 uL rev 10 uM primer
    • 1 uL DMSO
    • 21 uL nuclease-free water
    • 25 uL OneTaq 2X Master Mix with Standard Buffer
  • DpnI digested (0.5 μL DpnI per tube) Cas9-Lrz-SS for one hour at 37oC (heat block)
  • Nanodrop:
    • Before purification:
      • Cas9-Lrz-SS “A”: 449.4 ng/uL, 260/280: 1.51, 260/230: 0.66
      • Cas9-Lrz-SS “B”: 467.9 ng/uL, 260/280: 1.54, 260/230: 0.69
    • After purification:
      • Cas9-Lrz-SS “A”: 148.3 ng/uL, 260/280: 1.80, 260/230: 1.32
      • Cas9-Lrz-SS “B”: 56.0 ng/uL, 260/280: 1.84, 260/230: 1.29
  • Cas9-Lrz-SS product B was used in today’s Gibson reactions
  • Made Gibson reaction mixes with DsbA and Cas9-Lrz-SS (5:1 insert-to-backbone molar ratio)
    • Gibson assembly #1: DsbA insert with PCR-added homology
      • 0.89 uL Cas9-Lrz-SS PCR product from today
      • 0.23 uL DsbA insert with added homology PCR’ed today
      • 3.88 uL nuclease-free water
      • 5.00 uL Gibson master mix
    • Gibson assembly #2: DsbA insert gBlock
      • 0.89 uL Cas9-Lrz-SS
      • 1.67 uL DsbA gBlock
      • 2.54 uL nuclease-free water
      • 5.00 uL Gibson master mix
    • Negative control
      • 0.89 uL Cas9-Lrz-SS
      • 4.11 uL nuclease-free water
      • 5.00 uL Gibson master mix
  • Gibson assembly reactions were run at 50°C for one hour and transformed by Jordan and Sam

Tyler

  • Gel Screen of SS + Cas9
  • Aided with various steps of the cloning procedure (set up thermocyclers, did gibson calculations, etc.)