Team:Northwestern/07 28

Notebook

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

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