Team:Lethbridge Canada/notebook june

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Restrictions

  • Restricted (total 60µL DNA) K314100 in pSB1C3 with Spe1 and Pst1
  • Restricted (total 60µL DNA) Oxytocin in puc19a with Xba1 and Pst1
  • Restricted (total 60µL DNA) NEC1puc19a with Xba1 and Pst1
  • Set 12 µL aside from each reaction and gel extracted (GEX) the remaining 48µL
  • Combined heat killed restriction mixtures into ligation mix from NEB using NEB protocol, left at room temperature for 10 mins then heat killed for 20 mins at 80°C
  • K314100 – pSB1C3 (s,p), with: Oxytocin (x,p)
  • K314100 – pSB1C3 (s,p), with: NEC1 (x,p)
  • GEX K314100 – pSB1C3 (s,p), with: GEX Oxytocin (x,p)
  • GEX K314100 – pSB1C3 (s,p), with: GEX NEC1 (x,p)

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Transformation

  • Transformed ligations into 20µL high efficiency DH5α cells and 50µL lab made competent DH5α
    • K314100 – pSB1C3 (s,p), with: Oxytocin (x,p)
    • K314100 – pSB1C3 (s,p), with: NEC1 (x,p)
    • GEX K314100 – pSB1C3 (s,p), with: GEX Oxytocin (x,p)
    • GEX K314100 – pSB1C3 (s,p), with: GEX NEC1 (x,p)
  • Plated on CAM plates
  • Results: Only the 20µL non gel extracted DH5α E. coli high efficiency cells grew

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Picked Colonies

  • Date: June 4, 2013
  • Picked 10 colonies from each plate
  • Grew over night in LB and mini prepped
  • PCR-ed with VF2 and VR primers, no results as shown
  • Gel on June 4, 2013
  • Picked 6 Oxytocin colonies and 5 NEC1 colonies grew overnight in LB – mini prepped and restricted all colonies picked so far at E, P cute sites and ran on gel, results concluded as ligations did not work since a band is seen at ~500 bp size of the promoter construct K314100 but not with oxytocin or NEC1
  • Gel on June 4, 2013

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Picked Colonies

  • Date: June 10, 2013
  • Sent 10µL DNA with 5µL of 5µM VF2 primer to genewize
    • J23100-J06702
    • J23108-J06702
    • J23113-J06702
    • K314100-oxytocin neurophysin1
  • June 12th results arrived
    • J23100-J06702- positive for J06702 but no promoter found
    • J23108-J06702- positive for J06702 but no promoter found
    • J23113-J06702- positive for J06702 but no promoter found
    • K314100-oxytocin neurophysin1 – Positive results full sequence found

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Transformation of OXY into BL21

  • Date: June 13, 2013
  • Transform mini prepped plasmid DNA of K314100–Oxytocin Neurophysin1 in pSB1C3 into 15µL of Bl21 E.coli cells
  • Let grow over night at 37°C on CAM plates

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Restricted and Ligated and Transformed

  • Date: June 13, 2013
  • Restricted K314100 Spe1 and Pst1 and NEC1 at Xba1 and Pst1 for 1 hour at 37°C followed by 20 min heat kill at 80°C
  • Mixed restrictions in ratio of 10µL NEC1 and 2µL k314100 in pSB1C3 let sit in ligation mixture for 10 mins at room temperature followed by 20 min heat kill at 80°C
  • Transformed 2µL into 20µL of DH5α E.coli highly competent cells, incubated overnight at 37°C

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Picked Cells

  • Date: June 13, 2013
  • K314100–Oxytocin neurophysin 1 in pSB1C3 grew in Bl21 from last nights transformation, picked 4 colonies and grew up in 5ml of LB overnight at 37°C with shaking

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Ni-Sepharose Purification

  • Date: June 15, 2013
  • Grew 250mL culture of K314100 – Oxytocin neurophysin in psb1c3 overnight at 37°C with shaking (June 14)
  • Purified preprooxyphysin with Ni-Sapharose beads according to Wieden Protocol
  • Eluted overnight
  • Will confirm with tris-tricine PAGE

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Tris-Tricine PAGE

  • Date: June 18, 2013
  • Prepro-oxyphysin can be seen at the bottom of lanes 5 and 7
  • Lane Contents Volume
    1 Protein Molecular Weight Marker 20μL
    2 Cbf5-Nop10-Gar1 20μL
    3 Buffer A wash 30μL
    4 Buffer B wash 30μL
    5 Buffer E Wash 30μL
    6 S-30 s/N 30μL
    7 Buffer E – Concentrated Preprooxyphysin 30μL

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Anti-His Slot Blot

  • Date: June 21, 2013
  • Lane # Contents
    Lane 1: 100μL Oxytocin in 200μL TBS
    Lane 2: 200μL Oxytocin in 100μL TBS
    Lane 3: 250μL Oxytocin in 50μL TBS
    Lane 4: 300μL Oxytocin
    Lane 5: 25pM TruB-His positive control

    • We used a 25pM sample of the protein TruB with a histidine tag which is seen in lane 5. In lanes 1,2,3 and 4, faint bands are seen on the slot blot. This is indicative of the histidine tag attached to Oxytocin-NeurophysinI.

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