Team:Lethbridge Canada/notebook may

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<h1 class="notebook_entry_header">Ligations</h1>
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<li>Date: May 22, 2013</li>
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<li>12.5 ul dh20</li>
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<li>2ul t4 dna ligase buffer</li>
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<li>3ul DNA J06702</li>
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<li>1.5ul DNA J23100, J23108, J23113, J23114 in plasmid J61002</li>
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<li>1ul T4 dna Ligase</li>
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<li>Into the Thermo-cycler overnight at 16 degrees Celsius

Revision as of 00:31, 15 June 2013

Ligations

  • Name: Yoyo Yao
  • Date: May 1, 2013
  • Ligation of Parts – Upstream, Ligated at sites S & P:
    • J23100
    • J23113
    • J23108 +
    • J23108 –
    • All promoters already attached to Destination Plasmid J61002
  • Downstream, Ligated at sites X & P
    • J23100

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Restriction Digest

  • Name: Mackenzie
  • Date: May 4, 2013
  • 35µL DNA, 14µL MM, 0.5µL Spe1, 0.5µL Pst1
  • Master Mix
    Milliq 36µl
    BSA 45µl
    10x buffer 2 NEB 45µl

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1% Agarose Gel of Promoter Constructs

  • Name: Mackenzie
  • Date: May 4, 2013
  • Lane # Contents Volume
    1 1KB LADDER 6µL
    2 J23100_J61002 25µL
    3 J23100_J61002 25µL
    4 J23100_J61002 25µL
    5 J23100_J61002 25µL
    6 J23108_J61002 25µL
    7 J23108_J61002 25µL
    8 J23108_J61002 25µL
    9 J23108_J61002 25µL
    10 J23113_J61002 25µL
    11 J23113_J61002 25µL
    12 J23113_J61002 25µL
    13 J23113_J61002 25µL
    14 J23114_J61002 25µL
    15 J23114_J61002 25µL
    16 J23114_J61002 25µL
    17 J23114_J61002 25µL

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Gel Extraction

  • Date: May 4, 2013
  • J23108 J61002
  • J23113 J61002
  • J23114 J61002
  • J23100 J61002
  • Eluted 50 µL

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Transformation

  • Name: Mackenzie
  • Date: May 5, 2013
  • Tube # Part # Plasmid Volume of Competent Cells
    1 J23100+J06702 J61002 20µL
    2 J23108+J06702 J61002 20µL
    3 J23113+J06702 J61002 20µL
    4 J23108-J06702 J61002 20µL

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Restriction

  • Name: Chris Isaac
  • Date: May 7, 2013
  • Cut with ‘X’ and ‘P’
  • J06702 – RBS-mcherry-DT

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Gel Extraction

  • Name: Katie T. & Elaine B.
  • Date: May 7, 2013
  • J06702
  • Eluted at 30µL
  • Gel Extraction Done on May 7, 2013

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Gel Extracted Parts on Conformation Gel

  • 1µl of 1kb Ladder ng/0.5µg
  • 2µl DNA in all samples
  • Gel Extracted Parts on Conformation Gel Image
    Lane # Content
    1 Ladder
    2 J06702
    3 J23113–J61002 mc05/04/13mdy
    4 J23108–J61002 mc05/04/13
    5 J23100–J61002 mc05/04/13
    6 J23114–J61002 mc05/04/13
    7 Ladder
    8 J23100–J 61002 EK150413dmy
    9 J23108–J61002 EK150413
    10 J23113–J61002EK150413
    11 J23114 –J61002 EK150413

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Gel Extraction

  • Name: Carissa K.
  • Date: May 8, 2013
  • 2ul of J06702 mix with 4ul of each; J23100, J23108, J23113, J23114 all in plasmid J61002 (amp)
  • In the Thermo-cycler at 16 degrees Celsius overnight followed by heat kill.

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PCR of Ligation Mixture(PCR to verify insert in clone vector)

  • Name: Chris Isaac
  • Date: May 9, 2013
  • X7 Master Mix, Taq polymerase
  • J23100_J61002 (control)
  • J06702_pSB1AK2 (control)
  • J23100_J61002_J06702
  • J23108_J601002_J06702
  • J23113_J61002_J06702
  • J23114_J61002_J06702

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Restriction of PCR Ligations Gel Conformation

  • Name: Patrick O'
  • Date: May 13, 2013
  • Restriction of May 9th pcr ligation at E and P and ran on 1.0% Gel (refreshed ETH Bromide)
  • Ladder lane 2
  • 2space
  • J06702_pSB1AK2 (control)
  • 2spaces
  • J23100_J61002 (control)
  • spaces
  • J23100_J61002_J06702
  • J23108_J601002_J06702
  • J23113_J61002_J06702
  • J23114_J61002_J06702
  • Ladder
  • Conclusion: Ligations were not Successful
  • Restriction of PCR Ligations Gel Conformation Image

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Transformation of Ligations

  • Date: May 14, 2013
  • Transformed ligation of J06702 with various promoters in plasmid J61002 : J23100, J23108, J23113, J23114 From May 8, 2013.
  • Transformed 3ul ligation mix into 30ul of Dh5 alpha competent cells, used puc19 control. Heat shocked for 30second at 42 degrees Celsius added 200ul warm LB, incubated at 37 for 1 hour, Plated 100ul on amp LB Agar plates. Overnight at 37degrees Celsius.

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Ligation Round 2 of May 14th Gel Extracted Parts

  • 11ul dh20
  • 2ul t4 dna ligase buffer
  • 4ul DNA J06702
  • 2ul DNA J23100, J23108, J23113, J23114 in plasmid J61002
  • 1ul T4 dna Ligase
  • Into the Thermo-cycler overnight at 16 degrees Celsius.

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Picking Cells

  • Name: Yoyo, Amrinder, Patrick, Orion
  • Date: May 15, 2013
  • Put 50µL of Amp resistance into 50mL of Lb media
  • Flasks:
    • J23100_J61002
    • J23108_J61002
    • J23113_J61002
    • J23114_J61002
    • J06702_psb1ak2
  • Inserted tips of E. Coli into each flask

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Picking Cells

  • Name: Amrinder, Orion
  • Date: May 16, 2013
  • Put 5µL of Amp into 5mL of Lb media
  • Flasks:
    • J23100_J61002
    • J23108_J61002
    • J23113_J61002
    • J23114_J61002
    • J06702_psb1ak2
  • Inserted tips of E. Coli into each tube

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10 Fold Miniprep

  • Name: Isaac, Patrick
  • Date: May 16, 2013
  • Spun down 50ml of culture in falcon tubes
  • 5000xg twice for 2mins to collect cells
  • Resupended in 1ml solution1 2ml solution 2 and 3,5ml solution 3
  • Spun down at 6000xg for 6minutes to pellet cell debries
  • Eluted into 4 miniprep spin collumns twice to reach maximum binding effeciency
    • Combined eluted minipreps into one sample totalling 200µL
  • Mini prep of (50µL EB):
    • J23100_J61002
    • J23108_J61002
    • J23113_J61002
    • J23114_J61002
    • J06702_PSB1AK2
  • Mini-prepped with EZ-10 columns
  • Eluted 35µL
  • Purified DNA in Erin’s Box

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Agarose Gel

  • Name: Krista Fjordbotten, Yoyo Yao
  • Date: May 17, 2013
  • Restriction of:
    • 4μL DNA
    • 1μL Enzyme
    • 2.5μL Buffer
    • 17.5μL Water
    • J23100_J06702 J61002
    • J23108_J06702 J61002
    • J23113_J06702 J61002
    • J23114_J06702 J61002
    • J06702 J61002
  • Ladder: 2μL Ladder, 0.5μL Dye
  • Conformation Gel on May 17, 2013
  • Conformation of all 5 of May 16, 2013 minipreps
  • Well #(from left) DNA Parts Concentration
    2 ladder 2μL Ladder/0.5μL Dye
    4 J23100_J06702 J61002 25μL DNA/5μL Dye
    6 J23108_J06702 J61002 25μL DNA/5μL Dye
    8 J23113_J06702 J61002 25μL DNA/5μL Dye
    10 J23114_J06702 J61002 25μL DNA/5μL Dye
    12 J06702 J61002 25μL DNA/5μL Dye
    14 ladder 2μL Ladder/0.5μL Dye

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Miniprep

  • Name: Amrinder Grewal , Krista Fjordbotten
  • Date: May 17, 2013
  • Mini prep of (35µL EB):
    • J23100_J61002
    • J23108_J61002
    • J23113_J61002
    • J23114_J61002
    • Mini-prepped with EZ-10 columns
    • Eluted 35µL
    • Purified DNA in Erin’s Box
  • Inserted tips of E. Coli into each flask

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Glycerol Stock

  • Name: Amrinder Grewal , Krista Fjordbotten, Yoyo Yao, Isaac Ward
  • Date: May 17, 2013
  • Glycerol Stocked:
    • J23100_J61002
    • J23108_J61002
    • J23113_J61002
    • J23114_J61002

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Ligations