Team:Northwestern/09 13

Notebook

Tuesday, September 13th

Tasks:

Jordan

  • Worked on “Experiments” website content
  • Reread papers

Michelle

  • Nanodrop PCR purifications of GG parts:
    • M1: 64.2 ng/uL, 260/680: 1.89, 260/230: 1.41
    • M2: 53.9 ng/uL, 260/280: 1.84, 260/230: 1.24
    • M3: 17.8 ng/uL, 260/280: 1.89, 260/230: 1.21
    • M4: 43.8 ng/uL, 260/280: 1.86, 260/230: 1.61
    • M5: 32.4 ng/uL, 260/280: 1.77, 260/230: 0.88
    • G1: 66.5 ng/uL, 260/280: 1.90, 260/230: 2.05
    • G2: 63.1 ng/uL, 260/280: 1.91, 260/230: 1.89
    • G3: 66.6 ng/uL, 260/280: 1.80, 260/230: 1.14
    • G4: 44.6 ng/uL, 260/280: 1.89, 260/230: 1.89
  • Golden Gate dilutions:
    • M1: 129.9 fmol/uL, 6.16 uL dilute to 20 uL (40 fmol/uL)
    • M2: 109.0 fmol/uL, 7.34 uL dilute to 20 uL (40 fmol/uL)
    • M3: 36.0 fmol/uL, 11.11 uL dilute to 20 uL (20 fmol/uL)
    • M4: 88.59 fmol/uL, 9.03 uL dilute to 20 uL (40 fmol/uL)
    • M5: 65.54 fmol/uL, 12.21 uL dilute to 20 uL (40 fmol/uL)
    • G1: 134.5 fmol/uL, 5.95 uL dilute to 20 uL (40 fmol/uL)
    • G2: 127.6 fmol/uL, 6.27 uL dilute to 20 uL (40 fmol/uL)
    • G3: 134.7 fmol/uL, 5.94 uL dilute to 20 uL (40 fmol/uL)
    • G4: 90.21 fmol/uL, 8.87 fmol/uL dilute to 20 uL (40 fmol/uL)

Paul

  • PCR purified GFP1-4, m1-5 (see Michelle’s notes)
  • Designed primers for inserting Cas9 into T7 expression vector pET28a
  • Talked with grads about T7 expression line/vector

Sam

  • Auburn Gresham groceries
  • Started Auburn Gresham presentation
  • To do: update OneNote

Sara

  • Miniprep of Cas9-TorA overnight culture
  • Emailed the UNSW team asking about whether they thought 100 kDa filters would work with their protocol

Tasfia

  • PCR: Reran Tet-Lrz-GFP with same PCR conditions as 08.29.16
    • Three 50-uL reactions using “Tet plasmid miniprep” (conc’n 450 ng/uL)
    • One reaction with 45 ng template
    • Two reactions with 4.5 ng template
  • DpnI digested Tet-Lrz-GFP for one hour at 37°C on heat block
  • Ran gel screen on (non-DpnI digested) Tet-Lrz-GFP—faint bands that run at the correct size
    • 3 uL SybrSafe
    • 6 uL 1kb ladder + 3 uL 6X purple loading dye
    • In each sample, 1 uL 6X purple loading dye + 5 uL DNA
  • PCR purified Tet-Lrz-GFP
    • Gel Lanes 2+3: 15.2 ng/ul, 260/280: 1.89, 260/230: 1.17
    • Gel Lane 4: 13.3 ng/uL, 260/280: 1.83, 260/230: 1.17
  • In progress: making Auburn Gresham presentation slides
  • PCR: Reran Tet-LRz-GFP
    • Thermocycler 1 (right side); Q5 2X Master Mix
      • Two 50-uL reactions using 500 nM primer concentration in reaction mix
        • 0.5 uL Tet plasmid miniprep template (8.0 ng)
        • 2.5 uL 10 uM Tet_Lrz_GFP_FWD primer
        • 2.5 uL 10 uM Tet_Lrz_GFP_REV primer
        • 1 uL DMSO
        • 18.5 uL nuclease-free water
        • 25 uL Q5 HiFi 2X Master Mix
      • Two 50-uL reactions using 200 nM primer concentration in reaction mix
        • 1 uL Tet plasmid miniprep template (8.0 ng, 0.8 ng)
        • 1 uL 10 uM Tet_Lrz_GFP_FWD primer
        • 1 uL 10 uM Tet_Lrz_GFP_REV primer
        • 1 uL DMSO
        • 21 uL nuclease-free water
        • 25 uL Q5 HiFi 2X Master Mix
    • Thermocycler 2 (left side); OneTaq 2X Master Mix
      • Two 50-μL reactions using 200 nM primer concentration in reaction mix (as per usual)
        • 1 μL Tet plasmid miniprep template (8.0 ng, 0.8 ng)
        • 1 μL 10 μM Tet_Lrz_GFP_FWD primer
        • 1 μL 10 μM Tet_Lrz_GFP_REV primer
        • 1 μL DMSO
        • 21 μL nuclease-free water
        • 25 μL OneTaq 2X Master Mix with Standard Buffer