Thursday, July 28th
Sequencing Results:
Cas9 Gibson cultures A1, A2, A3, A5, B1, B2, B3, B5: Only the BBprefix_FWD primer gave us clean results, showing that the Cas9 was not successfully inserted into the linearized backbone. We realized that we used circular DNA in the Gibson assembly.
Tasks:
Jordan
- Transformed the GFP and mCherry ligations
- Ran all 9 tests, did a backbone-only ligation control, a plate with no DNA, and a control from the competent cell test (on Cam)
- Transformed 4 uL of plasmid DNA each, 1 uL for no DNA and comp cell controls
- Long ice incubation step because water bath wasn’t ready yet, ~45 minutes
Michelle
- Autoclaved
- LB media from 7/26/16 into smaller aliquot bottles
- Three beveled Erlenmeyers of 100 mL of LB for Cas9 expression cultures
- Pipette tips
- Eppendorf tubes
- Took a fridge inventory
- Vortexed all crashed out T4 Ligase Buffers
- Reran the linearizing Tet backbone PCR
- 0.5 μL of 10 μM forward primer
- 0.5 μL of 10 μM reverse primer
- 1.0 μL Tet in pSB1C3 template (~2100 bp)
- 12.5 μL 2X Q5 Master Mix
- 10.5 μL nuclease-free water
- 95°C (2:00) | 95°C (0:07), 63°C (0:10), 72°C (2:06) | 72°C (5:00)
- 25 μL reaction volume, 25 cycles
- Made T4 Ligase Buffer Aliquots (20 μL)
- Ran gel on Tet backbone PCR results
- Lane 1: 2 μL of 2-log Purple ladder + 6 μL of NEB Blue 6X loading dye
- Lane 3: 25 μL PCR reaction + 5 μL NEB Blue 6X loading dye
- RTW-PCR linearized Tet backbone to troubleshoot Master Mixes and Taq polymerase—Taq failure
Figure 1: PCR results. Lane 1: 2log ladder. Lane 2: No insert control. Lane 4: Master Mix, 0.5uL primers. Lane 6: Separate components, 0.5uL primers. Lane 8: Master Mix, 1.25uL primers. Lane 10: Separate components, 1.25uL primers
Sara
- Poured a gel for Michelle
- Analyzed sequencing
- Ran colony PCR
- 12.5 uL OneTaq MM
- 0.5 uL BB Vf Primer
- 0.5 uL BB Vr Primer
Shu
- Miniprep of mRFP in pSB1T3
- Made two stocks
- #1: 70.3ng/uL, 260/280:1.93, 260/230:1.93
- #2: 63.8ng/uL, 260/280:1.93, 260/230:1.95
- Ran a gel for GFP PCR product
- Gel extraction with Thush
Tasfia
- Ran gel and gel extraction for GFP and mCherry
