Team:Northwestern/09 12

Notebook

Monday, September 12th

Tasks:

Jordan

  • PCR’d GFP 1-4 and mCherry 1-5
    • 25 uL One Taq MM
    • 1 ul DMSO
    • 1 ul 10 mM fwd primer (Sara diluted these)
    • 1 ul 10 mM rev primer
    • 1 ul template (~1 ng)
    • 21 uL water
  • Conditions:
    • Initial denaturation, 95C/3:00
    • 5 cycles- 95/:30, 54/0:10, 72/0:43
    • 25 cycles- 95/:30, 68/0:10, 72/0:43
    • 72/5:00

Michelle

  • PCR for Cas9 lnrz ClyA
    • 18.5 uL water
    • 1 uL DMSO
    • 0.5 uL Cas9 1:3 miniprep
    • 2.5 uL Tet lnrz for Cas9 fwd (10 uM)
    • 2.5 uL ClyA rev (10 uM)
    • 25 uL 2X Q5 polymerase MM
    • Conditions: 95°C (3:00) | 95°C (0:30), 65°C (0:15), 72°C (2:40) | 72°C (5:00)
  • Cam plates:
    • 350 mL LB + 5.2528 g Bacto Agar
  • LB:
    • 10.0494 g tryptone
    • 5.0101 g yeast extract
    • 5.0015 g NaCl
    • 1 mL 1M NaOH
    • H2O to 1L

Paul

  • Submitted Sequencing:
    • 5 uL of DNA for all Cas9-Dsba’s (~450 ng)
    • 10 uL of DNA for all gRNA’s (~200-450 ng)
    • Tubes “1-12”= Cas9-Dsba 1-10, A, B
    • Tubes “13-22”= gRNA 1-10

Sam

  • Followed up on Field museum
  • Caught up OneNote
  • To do: Auburn Gresham powerpoint
  • To do (eventually): redesign survey

Shu

  • PCR of INP
  • Ran gel screening
  • Went over what people have done

Tasfia

  • PCR: Linearized tet backbone for GFP (same conditions and reaction mix as 08.29.16)
    • Two 50-uL reactions 45 ng (“Tet-Lrz-GFP A”)
    • 4.5 ng (“Tet-Lrz-GFP B”)
  • PCR: Adding homology to INP (re-running because on first run forgot to DpnI digest before purifying)
    • One 50-uL reaction
      • 1 uL INP miniprep template (8.6 ng)
      • 1 uL 10 uM INP_fwd primer
      • 1 uL 10 uM INP_rev primer
      • 1 uL DMSO
      • 21 uL nuclease-free water
      • 25 uL OneTaq 2X Master Mix with Standard Buffer
  • One INP PCR product—DpnI digested
  • PCR purified Tet-Lrz-GFP, Cas9-Lrz-ClyA, and [not-DpnI-digested] INP
    • Tet-Lrz-GFP A: 20.1ng/uL, 260/280: 1.93, 260/230: 1.46
    • Tet-Lrz-GFP B: 18.8ng/uL, 260/280: 1.99, 260/230: 2.49
    • Cas9-Lrz-ClyA: 125.2ng/uL, 260/280: 1.91, 260/230: 2.06
    • Questionable INP: 119.4ng/uL, 260/280: 1.89, 260/230: 1.94
  • PCR: Cas9-Res-Dig (eventually for parts submission)
    • One 50-uL reaction
      • 1 uL “Cas9 1:3” miniprep template (13 ng)
      • 1 uL 10 uM forward primer
      • 1 uL 10 uM reverse primer
      • 1 uL DMSO
      • 21 uL nuclease-free water
      • 25 uL OneTaq 2X Master Mix with Standard Buffer
  • DpnI (0.5 μL enzyme) digested for ~2 hours at 37°C, then left at room temperature overnight
  • Ran gel screen on unpurified, DpnI-digested Cas9-Res-Dig product
  • DpnI digesting (0.5 uL enzyme) at room temperature overnight
    • GFP 1-4 for golden gate
    • mCherry 1-5 for golden gate
    • INP with added homology (second INP PCR product of the day)
    • Cas9-Res-Dig (even though it was already going on the heat block for two hours before)

Tyler

  • Finished sequencing reactions, sent them off
  • Inoculated 5mL cultures of TorA-Cas9

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