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