Figure 1: Transformation positive control

Figure 2: Transformation negative control

Figure 3: Gibson assembly positive control

Figure 4: No enzyme negative control

Figure 5: gRNA + mRFP Gibson

Figure 6: INP + Cas9 Gibson

Figure 7: TorA + Cas9 Gibson

Jordan

• Wrote a lab note for the Experiment page
• Reserved room for another outreach discussion

Michelle

• Ran gel on Golden Gate reactions
  
  • The gel shows the inserts and backbone separately at their respective locations. It does not indicate success. M4 is more dim than expected.
• Streaked Delisa cells out on plates
  • JC8031 on no-antibiotic plate
  • JC8031-pClyA-GFP-His on Cam plate

Paul

• Pelleted 100 mL overnight expression cultures & put them in the freezer
• Emailed grad student listserv asking for help purifying proteins
• Looked through Cas9 purifying procedures

Sam

• Email more experts
• Researched questions in the research doc
• Started designing antibiotics forum

Sara

• Autoclaved some tips
• Lab notebook

Shu

• PCR mRFP with Tyler
  • 2 x 50µL reactions
  • 1µL DMSO
  • 1 µL mRFP backbone
  • 1 µL Tet Lnrz for Cas9 FWD (10µM)
  • 0.5 µL mRFP Reverse (10µM)
  • 21.5 µL water
  • Negative Control
  • 22 µL water
  • 1µL DMSO
  • 1 µL Tet Lnrz for Cas9 FWD (10µM)
  • 1 µL mRFP Reverse (10µM)
  • Conditions:
    
  • DpnI digested PCR products
    • 0.5 µL into each 50 µL
    • Incubated overnight at room temp
  • Nanodropped mRFP linearized: 125 ng/µL, 260/230: 2.30

Tyler

• PCR- Linearizing Cas9 for SS
  • 2 x 50µL reactions:
    • 21.5 µL water
    • 1 µL DMSO
    • 0.5 µL cas9 1:3
    • 1 µL SS_Lnrz_Fwd
    • 1 µL SS_Lnrz_Rev
    • 25 µL Taq Master Mix
  • Negative Control:
  • 22 µL water
  • 1 µL DMSO
  • 1 µL SS_Lnrz_Fwd
  • 1 µL SS_Lnrz_Rev
  • 25 µL Taq Master Mix
  • Conditions:
    
  • DpnI digest for 2 hours at 37°C
• PCR mRFP with Shu