Team:Northwestern/08 01

Notebook

Monday, August 1st

Tasks:

Jordan

  • Gibson reaction on 7.30 linearized tet backbone and Cas9 1+2
    • Insert:vector = 3:1
    • Backbone conc. 41 ng/uL
    • Used 50 ng backbone or 1.2 uL
    • Followed NEB kit protocol
    • Neg. Control- 1.2 ul vector + 8.8 ul water + 10 uL MasterMix
    • Positive control- 10 ul DNA provided in kit in 10 ul MasterMix
    • Incubated in water bath at 50 deg. 1 hour
  • Transformed Gibson product
    • One test condition, four controls
      • Actual Cas9 assembly—transformed 5 uL
      • Negative control Gibson with backbone only, should get no or few colonies—5 uL
      • Positive control from Gibson kit, should get colonies—5 uL
      • No DNA transformed, should not get colonies—1 uL
      • J04450 in pSB1C3- positive control, should get colonies—1uL
    • Plated 100 uL of each transformation

Michelle

  • Ran gel of Sara and Sam's 7.31 GFP and mCherry PCR
    • 50 uL PCR reaction per piece + 10 uL of 6X Blue Loading Dye
    • 25 uL loaded in each well
    • 2 ladders run per gel, 2uL of 2kb ladder + 6uL 6X Blue Loading Dye
    • Realized that Sara and Sam did not put template in, so the primer dimers on the gels made sense
  •  

  • Re-ran the PCR of the Tet Backbone Linearization for GFP/mCherry with Tyler
    • 2, 50µL reactions
      • 20 μL water
      • 1 μL DMSO
      • 2 μL tet backbone
      • 1 μL 10 μM fwd primer
      • 1 μL 10 μM rev primer
      • 25 μL OneTaq master mix
    • Negative Control (Water)
      • 22 μL water
      • 1 μL DMSO
      • 1 μL fwd primer
      • 1 μL rev primer
      • 25 μL OneTaq master mix
    • Same conditions as previous PCR, but increased the start and end annealing temperatures by 1°C
  • Took notes on Parallax scrolling, image transitioning in HTML and CSS—Started website

Paul

  • Ran a gel of GFP and mCherry PCR product with Sam
    • 4 wells per GFP/mCherry, 25 uL per well
    • mCherry seems to have worked, GFP did not

     

  • Looked over Cas9 Signaling Sequence primers: lots of primer dimers-probably easier/better to synthesize whole things
  • Investigate other SS
  • Feedback during group discussion
  • Got caught up from weekend work

Sam

  • Autoclaved TAE
  • PCR of GFP/mCherry
    • 25 uL OneTaq
    • 1 uL diluted 10 mM f primer
    • 1 uL diluted 10 mM r primer
    • 1 uL GFP/mCherry
    • 21 uL dH20
    • 1 uL DMSO
    • 95°C (2:00) | 95°C (0:07), 51°C (0:10), 72°C (0:43) | 72°C (5:00)
  • DpnI digest: 1 uL of DpnI added to each of the 50uL tubes. Incubated in the 37 for 4 hours
  • Ran gel on GFP/mCherry PCR with Paul

Sara

  • Ran a PCR of GFP and mCherry to put on the GG ends with Sam
  • Talked to Patrick to get more Dpn1 to use on the GFP mCherry PCR
  • Learned that we’ve been using too much Dpn1, and Patrick suggested using 1 uL per 100 uL tube

Tasfia

  • PCR Tet Backbone Linearization for GFP/mCherry with Tyler
    • Reactions
      • 10 μL water
      • 0.5 μL DMSO
      • 1 μL tet backbone
      • 0.5 μL 10 μM fwd primer
      • 0.5 μL 10 μM rev primer
      • 12.5 μL OneTaq master mix
    • Negative control for 25-μL reaction (1 tube)
      • 11 μL water
      • 0.5 μL DMSO
      • 0.5 μL fwd primer
      • 0.5 μL rev primer
      • 12.5 μL OneTaq master mix
  • DpnI digest on PCR product (added 1 μL DpnI in each reaction tube)
  • Ran a gel on the PCR product of Tet Backbone Linearization for GFP/mCherry
    • Two 8-μL ladders on each side of gel; 1:3 purple 2-log ladder to 6X blue loading dye
    • Each well had ~28 μL PCR product + loading dye (~26 PCR product + ~5.2 μL 6X blue loading dye)
    • 95 V
    • Ran for ~1 hour

Tyler

  • PCR Tet Backbone Linearization for GFP/mCherry with Thush
  • Gel on Tet linearization with Thush
  • PCR of the Tet Backbone Linearization for GFP/mCherry with Michelle
  • Reviewed SS cas9 parts

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