Jordan

• Updated inventory I’ve used this week
• Read GG protocol again
• Discovered with Paul that the incubator has been unplugged for at least 3 days

Michelle

• GFP Gel extraction
  • GFP1:
    • 0.1541 g dissolved in 0.4623 mL GEX
    • 0.1838 g dissolved in 0.5514 mL GEX
  • GFP2: 0.2071 g dissolved in 0.6213 mL GEX
  • GFP3: 0.1640 g dissolved in 0.4920 mL GEX
  • GFP4: 0.1186 g dissolved in 0.3558 mL GEX
  • 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
  • Results:
    • GFP1: 72.2 ng/μL, 260/280: 1.93, 260/230: 1.61
    • GFP2: 86.0 ng/μL, 260/280: 1.87, 260/230: 1.40
    • GFP3: 40.0 ng/μL, 260/280: 1.84, 260/230: 1.00
    • GFP4: 7.1 ng/μL, 260/280: 1.92, 260/230: 1.19
• Ran GFP4 PCR again:
  • 25 μL 2X OneTaq Mastermix
  • 1 μL 10 μM forward primer
  • 1 μL 10 μM reverse primer
  • 1 μL DMSO
  • 1 μL GFP template
  • 21 μL nfH₂O
  • 95°C (2:00) | 95°C (0:07), 51°C (0:10), 72°C (0:43) | 72°C (5:00)
• Ran gel on GFP4 re-attempt
  • Ladders: 2 μL NEB Purple 2-log ladder + 6 μL NEB 6X Blue Loading Dye
  • Loaded: 25 μL PCR reaction + 5μL NEB 6X Blue Loading Dye
  
• Gel extracted GFP4
  • Lane 2: 0.0665 g dissolved in 0.1995 mL GEX
  • Lane 4: 0.1378 g dissolved in 0.4134 mL GEX
  • Lane 6: 0.1515 g dissolved in 0.4545 mL GEX
  • Lane 8: 0.1568 g dissolved in 0.4704 mL GEX
  • Used Epoch kit with iGEM modified protocol
  • Results: 106.6 ng/μL, 260/280: 1.93, 260/230: 1.72
• Made dilutions for Golden Gate:
  • Tet backbone (20 fmol, 25 µL)
    • 8.70 µL of 37.3 ng/µL stock to 12.5 µL
    • 8.82 µL of 36.8 ng/µL stock to 12.5 µL
    • Because not enough of either to do it in one step
  • GFP 1 (40 fmol, 15 µL)
    • 4.11 µL of 72.2 ng/µL stock to 10 µL
  • GFP 2 (40 fmol, 10 µL)
    • 2.30 µL of 86.0 ng/µL stock to 10 µL
  • GFP 3 (40 fmol, 10 µL)
    • 4.94 µL of 40.0 ng/µL stock to 10 µL
  • GFP 4 (40 fmol, 10 µL)
    • 1.86 µL of 106.6 ng/µL stock to 10 µL
  • mCherry 1 (40 fmol, 10 µL)
    • 3.02 µL of 65.5 ng/µL stock to 10 µL
  • mCherry 2 (40 fmol, 10 µL)
    • 1.83 µL of 108.4 ng/µL stock to 10 µL
  • mCherry 3 (40 fmol, 10 µL)
    • 2.15 µL of 92.1 ng/µL stock to 10 µL
  • mCherry 4 (40 fmol, 10 µL)
    • 3.59 µL of 55.1 ng/µL stock to 10 µL
  • mCherry 5 (40 fmol, 10 µL)
    • 4.34 µL of 45.6 ng/µL stock to 10 µL
  • TorA (40 fmol, 50 µL)
    • First diluted 100µM stock to 1µM stock (0.5µL of 100µM to 50µL)
    • Second diluted to 40 fmol (2 µL of 1µM to 50µL)
  • YcdO (40 fmol, 50 µL)
    • First diluted 100µM stock to 1µM stock (0.5µL of 100µM to 50µL)
    • Second diluted to 40 fmol (2 µL of 1µM to 50µL)
  • AmiA (40 fmol, 50 µL)
    • First diluted 100µM stock to 1µM stock (0.5µL of 100µM to 50µL)
    • Second diluted to 40 fmol (2 µL of 1µM to 50µL)

Paul

• DpnI digest pSB1T3 linearized BB

Sara

• Updated the lab notebook and also added a few protocols

Tasfia

• Human practices
  • Released queries for experts in antibiotic resistance via journalists’ online source database ~ awaiting responses but it could be awhile
  • Sent emails to hopefully interview experts (clinical nurses, because we haven’t touched on them yet, but I plan to do more research on antibiotic resistance before I reach out to Medical Advisory Committees)
  • Updated document called “Planning/Brainstorming” with ideas, snippets of antibiotic resistance in current events, brainstormed methods

Tyler

• Ordered Tat, Sec, Tat/Sec SS w/Cas9 homology, finalized DsbA with Cas9 homology design and ordered that too
• Sponsor comm w/Bio-Rad, picked up western blot gels
• Researched translocation pathways, chose optimal sequences