Team:Northwestern/08 02

Notebook

Tuesday, August 2nd

Tasks:

Michelle

  • mCherry Gel Extractions
    • mCherry 1 gel parts:
      • 0.3712 g, dissolved in 1.1140 mL of GEX buffer
      • 0.3501 g, dissolved in 1.0501 mL of GEX buffer
    • mCherry 2 gel parts:
      • 0.3139 g, dissolved in 0.9417 mL of GEX buffer
      • 0.2385 g, dissolved in 0.7155 mL of GEX buffer
      • 0.3169 g, dissolved in 0.9507 mL of GEX buffer
    • mCherry 3 gel parts:
      • 0.3782 g, dissolved in 1.1346 mL of GEX buffer
      • 0.3702 g, dissolved in 1.1106 mL of GEX buffer
      • 0.3138 g, dissolved in 0.9414 mL of GEX buffer
    • mCherry 4 gel parts:
      • 0.2850 g, dissolved in 0.855 mL of GEX buffer
      • 0.3295 g, dissolved in 0.9885 mL of GEX buffer
    • mCherry 5 gel parts:
      • 0.3405 g, dissolved in 1.0215 mL of GEX buffer
      • 0.3521 g, dissolved in 1.0563 mL of GEX buffer
      • 0.3257 g, dissolved in 0.9771 mL of GEX buffer
    • Used Epoch’s kit w/ team procedure modifications:
      • Washed with WS twice
      • Let stand for each wash for 1 minute
      • Let stand before spinning for elution for 5 minutes
      • Eluted in 20 μL of n.f. water instead of elution buffer
  • Autoclaved 1 mL tips
  • Autoclaved 250 mL of LB for Cam plates
    • 0.25 mL of 1000X (35 mg/mL) Cam
    • 1 sleeve poured
  • HTML & CSS for the website

Paul

  • Looked into DsbA as a SS for Cas9
  • Transformed Gibson product:
    • 50µL competent cells, 5 µL of Gibson product, 1µL of positive control (psb1c3-mrfp), 1µL of negative control (religation of tet backbone)
    • -waited ~30 minutes
    • 45 second heat shock
    • 450µL of SOC added to each tube
    • Incubated for an hour
    • Plated 20 and 200µL plates for each sample
    • Controls:
      • Neg: Tet linearized BB+ligase 15 min (used 41 ng/uL BB)
      • Pos: pSB1C3_J04450 (Transformation Efficiency Test kit: 50 pg/uL)
  • Looked at gels of Linearized Tet BB for GFP and GFP

Sara

  • Ran test gel of 08.01.16 GFP PCR:
    • 2 uL of Sybr Green, ~30 mL 1% agarose
    • 5 uL PCR reaction per piece + 1 uL of 6X Blue Loading Dye
    • 6 uL loaded in each well
    • 2 NEB 2 kb ladders run per gel, 2uL of 2kb ladder + 6uL 6X Blue Loading Dye
  • Dpn1 digest of 08.01.16 GFP PCR with Shu
    • 0.5 uL Dpn1 in each 50 uL tube
    • Incubated for 2 hours at 37°C
  • Lab notebook labelling and organizing
  • Read up on golden gate procedures
  • Emailed Patrick

Shu

  • PCR of GFP again with Sara
  • Ran a final gel and gel extracted the GFPs
    • Ran a final gel on the digested product, 10uL loading dye in each product, 25uL in each well

Tasfia

  • Ran gel of 8.1 PCR Tet Backbone Linearization for GFP/mCherry
  • Gel extraction of Tet Lrz for GFP
    • Lane 1: 219 mg gel excised, dissolved in 657 μL GEX buffer
    • Lane 2: 300 mg gel excised, dissolved in 900 μL GEX
    • Lane 3: 271 mg gel excised, dissolved in 813 μL GEX
    • Lane 4: 219 mg gel excised, dissolved in 657 μL GEX
    • Lane 4: 219 mg gel excised, dissolved in 657 μL GEX
    • Used EPOCH’s iGEM-modified protocol for purification
    • Let product sit for 1-2 minutes between washes
    • Washed with WS buffer twice
    • Let product sit for 5 minutes for more optimal elution
    • Used nuclease-free water instead of elution buffer
    • Results: (4 tubes; data has been averaged from four NanoDrop runs per tube)
      Tube Concentration (ng/uL) 260/280 260/230
      1 36.8 1.86 1.12
      2 37.3 1.92 1.51
      3 27.4 1.90 1.34
      4 25.0 1.94 0.65

Tyler

  • Ligation of Linearized Tet backbone
    • 15.8µL water
    • 1.2 µL of backbone
    • 2µL of ligase buffer
    • 1µL of ligase
    • 15 minute wait 2:38-2:53
  • Transformation of Gibson Product with Paul
  • Looked into dsba SS, designed part
  • Began designing split Cas9 primers for gibson assembly (dimerized Cas9)
  • 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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