Team:Northwestern/08 17

Notebook

Wednesday, August 17th

Results:

Sequencing:

saCas9 Gibson Assembly was correctly assembled.

Transformation:

The Gibson assemblies of signal sequences into Cas9 were unsuccessful.

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

Tasks:

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

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