Team:Northwestern/08 16

Notebook

Tuesday, August 16th

Results:

The only transformation that was successful was the positive transformation control from the NEB competent cells box.

Tasks:

Jordan

  • Loaded linearized Cas9 gel
  • Ran linearized for SS cas9 backbone gel
  • Ran gel at 95 V for ~1 hr 20
  • Transformed Gibson reactions
    • 3 ul of each, 3 ul of 50 pg/ul Competent Cell test RFP, 3 ul nf water
    • Thawed cells 15 minutes before transformation
    • On ice for slightly more than 30 minutes
    • Heat shocked by Sam
    • Added 200 uL of SOC (maybe slightly less chilled because working near flame?)
    • Incubated 1 hour 15 minutes
    • Plated 250 uL

Michelle

  • Made glycerol stocks of Cas9 grown w/ antibiotic (colony A) and Cas9 grown without antibiotic
    • 500 uL of 50% glycerol
    • 500 uL of cell culture
  • Miniprepped Cas9 cultures and Tet backbone cultures
    • Cas9 grown in antibiotic (colonies A, B, C)
    • Cas9 grown without antibiotic
    • Tet backbone (colonies A, B, C)
    • Nanodrop results:
      • Tet plasmid colony A: 450.9 ng/uL, 260/280: 1.88, 260/230: 2.52
      • Tet plasmid colony B: 107.4 ng/uL, 260/280: 1.76, 260/230: 1.16
      • Tet plasmid colony C: 125.3 ng/uL, 260/280: 1.93, 260/230: 3.07
      • Cas9 w/ antibiotic colony A: 147.1 ng/uL, 260/280: 1.63, 260/230: 0.78
      • Cas9 w/ antibiotic colony B: 218.1 ng/uL, 260/280: 1.58, 260/230: 0.72
      • Cas9 w/ antibiotic colony C: 101.0 ng/uL, 260/280: 1.66, 260/230: 0.80
      • Cas9 w/o antibiotic: 42.4 ng/uL, 260/280: 1.83, 260/230: 1.40
  • Made LB
    • 10.0190 g tryptone peptone
    • 5.0098 g yeast extract
    • 5.0065 g NaCl
    • 1 mL 1 N NaOH
    • Used 100 mL for expression culture
    • Used 200 mL for plates
      • Added 3.0043 g Bacto agar
      • Poured 200 mL of Cam plates (0.2 mL Cam 1000X added)
  • Gel extracted Tet linearized for GFP
    • Part 1 + 2: 0.2783 g (0.8349 mL GEX buffer added)
    • Nanodrop results: 14.2 ng/uL, 260/280: 2.02, 260/230: 0.87
  • Gel extracted Tet linearized for gRNA
    • Part 1: 0.3604 g (1.0812 mL GEX buffer added)
    • Part 2: 0.3837 g (1.1511 mL GEX buffer added)
    • Nanodrop results: 125.7 ng/uL, 260/280: 2.02, 260/230: 1.94

Sam

  • Nanodrop data
    • Suspensions of gene blocks were 5 ng/uL instead of 10 and had awful ratios. 3 to 5 = 260/280, -0.4ish = 260/230.
      • Kelly said to trust the nanodrop and run the Gibson assuming they’re actually 5 ng/uL
    • Something was 5500 ng/uL. Ratios were good
      • Solution: dilute with nfH2O
  • Worked on the phone call script with Jordan
    • No response from yesterday’s call. Called more numbers.
  • Performed gel extraction of linearizing Cas9 gel.
  • Accidentally forgot to let elution water sit for five minutes. Concentration is still fine. 260/230 is 0.8

Sara

  • Updated the lab notebook and nagged people about writing down the things they’ve done
  • Checked OD of the cas9 cultures we’re growing up

Shu

  • Gibson Reactions
    • Cas9-TorA (vector:insert=1:5)
      • 0.64uL backbone
      • 1.11uL TorA gblock
      • 3.25uL water
    • 5uL master mix Cas9-INP (vector:insert=1:3)
      • 0.64uL backbone
      • 0.65uL diluted INP
      • 3.71uL water
    • 5uL master mix mRFP backbone-gRNA
      • 1.05uL diluted mRFP
      • 1.66uL template cutting gRNA gblock
      • 2.29uL water
      • 5uL master mix
    • Positive control
      • 5uL positive control
      • 5uL master mix
    • Negative control
      • 0.64uL backbone
      • 1.11uL TorA gblock
      • 8.25uL water
  • Re-nanodrop linearized Cas9: concentration: 39ng/uL

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
  • Ran Calculations for Gibson
  • Began loading gel for PCR product, let Jordan finish
  • Nanodropped some of our gblocks/troubleshot DNA concentrations
  • Took a screwdriver to the ice in the fridge

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