Team:Newcastle/Notebook/LabBookV1/July

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July 1st

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July 2nd

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July 3rd

    Fim Switch
  • Marcia + Lais
    • Digest ran for K1632013 (MP17) and K137058 (MP18)
      37C 1 hour, 64C 20 minutes, 4C rest
      2 copies of each sample digested
      17 + 18 - Arac/FimE
      22 + 21 - FimS/GFP

      For ligation, samples were quantifies on the nanodrop
      MP17 - 264.2 ng/µl
      MP18 - 306.8 ng/µl
      MP22 - 143.9 ng/µl
      MP21 - 117.5 ng/µl


      Competent cell test kit Calculating cell efficiency
      Ran gel + cut bands

      MP17 - 0.193 g
      MP18 - 0.205 g
      MP21 - 0.202 g
      MP22 - 0.292 g

      Gel purified using the Qiagen purification kit

      Competent cells testing kit using the iGEM protocol
       Controls
      • DH5α + H20 in LB
      • DH5α + H20 on cm
      • DH5α + GFP on cm
      • DH5α + mRFP on amp
      As well as transforming K1334H

July 4th

    Fim Switch
  • Ansh + Lais
    • Counting cell efficiency
      ---10 50 100
      1 - 2 20 46
      2 - 9 37 73
      3 - 6 36 34


  • Lais, Ansh, Marcia + Sophie
    • Transformation

July 5th

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July 6th

    Fim Switch
  • Marcia, Sophie + Ansh
    • Digestion
      MP17
      • 2 µl buffer (cutsmart)
      • 3.8 µl DNA
      • 1 µl Pst1
      • 1 µl Spe1
      • 12.2 µl water

      MP21
      • 2 µl buffer (cutsmart)
      • 8.5 µl DNA
      • 1 µl Pst1
      • 1 µl Xba1
      • 7.5 µl water

      Incubate at 37C 1 hour, 64C 20 minutes, 4C rest

July 7th

    Fim Switch
  • Sophie + Marcia
    • Used E-Gel to purify digest
      20 µl of MP17 and MP21, no dye added
      25 µl water in empty wells
      1kb plus ladder - 10 µl in M lane

      MP17 in lane 4
      MP21 in lane 5

      Digestion wasn't successful, MP21 was smeared in the gel
      Extracted MP17 but not MP21

July 8th

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July 9th

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July 10th

    Fim Switch
  • Lais, Marcia + Sophie
    • Digestion
      Digested MP18 and MP22

      Ran gel after digest and cut the band
      MP18 - 0.255 g
      MP22 - 0.274 g

      Gel extraction using Qiagen extraction kit

      Ligation
      Ligating at 1:1, 1:3 and 1:5 ratios

      1:1
      • 2 µl buffer x10
      • 1 µl vector (MP18)
      • 1 µl insert (MP22)
      • 1 µl T4 ligase
      • 15 µl water

      1:3
      • 2 µl buffer x10
      • 1 µl vector (MP18)
      • 3 µl insert (MP22)
      • 1 µl T4 ligase
      • 13 µl water

      1:5
      • 2 µl buffer x10
      • 1 µl vector (MP18)
      • 5 µl insert (MP22)
      • 1 µl T4 ligase
      • 11 µl water

      Set overnight (16 h) at 16C

July 11th

    Fim Switch
  • Lais, Marcia + Sophie
    • Transformed cells
      Used Bradley's protocol

July 12th

    Fim Switch
  • Whats this one?
    • 10 ml -> LB final volume
      CAM 2.5 mg/ml -> 12.5 µg/mg

      µg 12.5 x 10 = 2500 x V
      V = 0.05 ml
      = 50 µl

      10 ml -> LB
      50 µl -> Anti B -> CM
      1 colony


      Colony from 1:3 plate transformation started

July 13th

    Fim Switch
  • Marcia + Lais
    • Miniprepped Bradley's stuff, FimE and Lais' promoters
      Following Qiagen protocol

July 14th

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July 15th

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July 16th

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July 17th

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July 18th

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July 19th

  • Description
  • Declan
    • Transformation of the following constructs into DH5a cells:

      • Detector module - Successful
      • Connector 1 (detector cell) - Unsuccessful – Retransform on 20/07/17
      • Connector 1 (processing cell) - Successful
      • Connector 2 (processing cell) - Successful
      • Connector 2 (output cell) - Successful
      • Output module - Successful

      Positive and negative controls as expected

July 20th

  • Description
  • Lais
    • Re-transformation of Connector 1 (detector cell) construct
      – Successful

July 21st

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  • Sophie
    • Harvesting Cells and Prep.
      200 ml overnight culture of cells harvested following Bradley's cell free extract preparation protocol

July 22nd

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July 23rd

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July 24th

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  • Sophie
    • Continued from 21/07/17
      Followed the remainder of Bradley's cell free protocol from 21/07/17

July 25th

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July 26th

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July 27th

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July 28th

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July 29th

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July 30th

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July

Mo Tu We Th Fr Sa Su
26 27 28 29 30 1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31 1 2 3 4 5 6

What We Got Up To In July

Summary