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