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
- 2 x 50µL reactions:
- PCR mRFP with Shu
