Team:Lethbridge Canada/notebook april

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<li class="notebook_link_title"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march">March</a></li>
<li class="notebook_link_title"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march">March</a></li>

Revision as of 19:56, 19 June 2013

Restriction - Ligation

  • Names: Yoyo Y, Wesley M
  • Date: April 2, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • J06702 - mCherry
  • J61002 - RFP Reporter
  • Promoters: J23113, J23108, J23114, J23100
  • Cut Promoters at sites E & S
  • Cut mCherry at sites X & P
  • Cut Destination Plasmid E1010_pSB1C3 sites E&P
  • Promoter Master Mix
    • MilliQ: 66.25µL
    • BSA: 1.25µL
    • Buffer #2: 12.5µL
    • DNA: 8µL
    • Add 0.5µL EcoR1 and 0.5µL Spel to 16µL MM
  • J06702 Master Mix
    • MilliQ: 66.25µL
    • BSA: 1.25µL
    • Buffer #2: 12.5µL
    • J06702: 40µL
    • Add 0.5µL Xbal and 0.5µL Pst1 to 24MM
  • pSB1C3 Master Mix
    • MilliQ: 66.25µL
    • BSA: 1.25µL
    • Buffer #2: 12.5µL
    • E1010 pSB1C3: 40µL
    • Add 0.5µL EcR1 and 0.5µL Pst1 to 24MM
  • Incubated at 37°C for 1 hour
  • Heat killed enzyme for 20 mins at 65°C

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Ligations

  • Name: Yoyo Y
  • Date: April 2, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • Tube 1 - J23100_J06702_pSB1C3
  • Tube 2 - J23108_J06702_pSB1C3
  • Tube 3 - J23113_J06702_pSB1C3
  • Tube 4 - J23114_J06702_pSB1C3
  • Left overnight at 16°C
  • Agarose Gel of Digested Parts 1%
  • Lane # Contents Volume
    1 1 kb DNA Ladder 6µL
    2 E1010 pSB1c3 #4 15µL
    3 E1010 pSB1c3 #3 15µL
    4 E1010 pSB1c3 #2 15µL
    5 E1010 pSB1c3 #1 15µL
    6 J06702 #4 15µL
    7 J06702 #3 15µL
    8 J06702 #2 15µL
    9 J06702 #1 15µL
    10 J23100 15µL
    11 J23108 15µL
    12 J23113 15µL
    13 J23114 15µL

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Transformation

  • Name: Fiona S, Chris I, Yoyo Y
  • Date: April 3, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • Antibiotic: CAM
  • Part Number and Plasmid: J23100_J06702_pSB1C3, J23108_J06702_pSB1C3, J23113_J06702_pSB1C3, J23114_J06702_pSB1C3
  • Results: TBA

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Re-Plating Transformation

  • Names: Chris I, Fiona S
  • Date: April 4, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • Part Number and Plasmid: J23100_J06702_pSB1C3, J23108_J06702_pSB1C3, J23113_J06702_pSB1C3, J23114_J06702_pSB1C3
  • Antibiotic CAM

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Miniprep of ON Cultures

  • Names: Chris I, Yoyo Y
  • Date: April 5, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • Miniprepped J23108_pSB1A2, J23113_pSB1A2, J23114_pSB1A2, J06702_pSB1AK2, used 50µL of Elution Buffer

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UV Spectroscopy DNA Concentrations

  • Names: Chris I, Yoyo Y, Elaine B
  • Date: April 5, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • A260 A280 A260/A280 DNA Concentration
    at 50-fold
    dilution ug/mL
    J23108 0.1017 0.0092 11.05435 254.25
    J23113 0.1289 0.0338 3.813609 322.25
    J23114 0.135 0.0397 3.400504 337.5
    J06702-1 0.13 0.0373 3.485255 325

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Miniprep From Ligation

  • Names: Shikhar M, Krista F, Austin K
  • Date: April 6, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • Miniprepped J23108_J06702_pSB1C3, J23113_J06702_pSB1C3, J23100_J06702_pSB1C3 used 50µL of Elution Buffer
  • Note: left plates to grow longer, need DNA sequenced.

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PCR

  • Name: Erin K
  • Date: April 7, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSB1AK2
  • Separately added 2µL of appropriate DNA and 0.2µL of Pfu Polymerase to 17.8µL MM
  • Agarose Gel confirmation of PCR: Success
  • MM 5x Reagent Volume(µL)
    MilliQ H2O 67
    10X Pfu Buffer with MgSO4 20
    10mM dNTPs 2
    VF2 5
    V4 5

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Restriction Digestion of PCR Parts

  • Name: Erin K
  • April 8, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • 2 samples of J06702_pSB1AK2: Added 0.5µL XbaI and 0.5µL PstI to 24µL MM
  • J06702 MM 2x Volume (µL)
    MIlliQ 29.5
    BSA 0.5
    NEB 10x Buffer 2 5
    J06702_pSB1AK2 10
  • 2 samples of TAT-E1010_pSB1C3: Added 0.5µL EcoRIand 0.5µL PstI to 24µL MM
  • TAT-E1010_pSB1C3 MM 2x Volume (µL)
    MIlliQ 12.5
    BSA 0.5
    NEB 10x Buffer 2 5
    TAT-E1010_pSB1C3 30
  • 2 samples of each of the 3 promoters: Added 5µL appropriate DNA and 0.5µL EcoRI and 0.5µL SpeI to 16µL MM
  • Promoter MM 7x Volume (µL)
    MIlliQ 95.75
    BSA 1.75
    NEB 10x Buffer 2 17.5

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3% Agarose Gel of Digested PCR Parts

  • Names: Wesley M, Riley M, Krista F
  • Date: April 8, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • Parts: J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSB1AK2
  • Results: Unsuccessful
    Lane # Contents Volume(µL)
    1 Ladder 100bp 5
    2 J23108 29
    3 J23108 29
    4 Blank N/A
    5 J23113 29
    6 Blank N/A
    7 Blank N/A
    8 Blank N/A
    9 J23113 29
    10 Blank N/A
    11 J23114 29
    12 Blank N/A
    13 J23114 29

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Gel Extraction

  • Names: Wesley M, Riley M, Krista F
  • Date: April 8, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Biobasic Kit: Eluted with 30µL elution buffer

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Repeat PCR

  • Name: Elaine B, Chris I, Fiona S, Wesley M, Riley M
  • Date: April 9, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSB1AK2
  • Separately added 2µL of appropriate DNA and 0.2µL of Pfu Polymerase to 17.8µL MM
    • \
  • MM 5x Reagent Volume (μL)
    MIlliQ 67
    10X Pfu Buffer with MgSO4 2
    10mM dNTPs 2
    VF2 5
    V4 5

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Picking Cells

  • Names: Chris I, Elaine B, Fiona S, Wesley M, Riley M,
  • Date: April 9, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • Picked: J23113_J61002, J23108_J61002, J23114_J61002, J23100_J61002, J06702_pSB_1AK2
  • 5mL cultures, 5µL Amp. grown overnight at 37°C

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Mini Prep

  • Names: Yoyo Y
  • Date: April 10, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • Mini Prep of (x2 of 35µL): J06702_pSB1AK3, J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002
  • Mini-prepped with EZ-10 columns
  • Eluted 35µL
  • Purified DNA in HS iGEM Kit #1

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Restriction

  • Names: Dylan S
  • Date: April 10, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • Add 33µL DNA to 17µL MM
  • 17µL MM to promoter tubes
  • 33µL DNA to promoter tubes
  • Same procedure for J06702_pSB1AK3
  • Incubated at 37°C for 2 hours
  • Heat killed at 65°C for 20 minutes
  • Promoter MM 50μL rxn x4 Volume (μL)
    MIlliQ 44
    BSA 20
    NEB 10 x Buffer 2 20

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Agarose Gel of PCR 1%

  • Result: Successful

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Miniprep

  • Names: Dylan S, Patrick O
  • Date: April 11, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • Mini prep of (35µL EB): J06702_pSB1AK3, J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002
  • Mini-prepped with EZ-10 columns
  • Eluted 35µL
  • Purified DNA in HS iGEM Kit #1

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Restriction Digestion

  • Names: Wesley M, Yoyo Y
  • Date: April 12, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • Restriction of E1010_pSB1C3 at sites E & P
  • 50µL rxn
  • Add 30µL DNA to 20µL MM
  • Added 0.5µL EcoR1 & Pst1
  • Incubated 2 hours at 37°C
  • Heat killed 20 minutes at 65°C
  • MM x 2.5 Volume (μL)
    MIlliQ 22.5
    10x Buffer 2 (NEB) 12.5
    BSA 12.5

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PCR and Cell Reculturization

  • Names: Patrick O, Shikhar M
  • Date: April 12, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • Reculturization of: J06702_pSB1AK3, J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002
  • PCR: Master mix created with: Annealing temperature: 55.6°C
  • MM for Promoters (x5) Conditions for tube "a"
    MIlliQ 67 μL MIlliQ 13.4 μL
    10X Pfu Buffer + MgSO4 20 μL 10X Pfu Buffer + MgSO4 4 μL
    10mM dNTPs 2 μL 10mM dNTPs 0.4 μL
    VF2 5 μL VF2 1 μL
    VR 5 μL VR 1 μL
    Pfu polymerase 0.2 μL Pfu polymerase 0.2 μL
    DNA part 2 μL

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Miniprep

  • Names: Amrinder G, Steven T
  • Date: April 12, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • Mini prep of (35µL EB): J06702_pSB1AK3, J23100_J61002, J23108_J61002 ,J23113_J61002, J23114_J61002
  • Mini-prepped with EZ-10 columns
  • Eluted 35µL
  • Purified DNA in HS iGEM Kit #1

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Miniprep

  • Names: Fiona S, Katie T
  • Date: April 14, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • Mini prep of (35µL EB): J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSB1AK2
  • Mini-prepped with EZ-10 columns
  • Eluted 35µL

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1% Agarose Gel

  • Names: Fiona S, Katie T
  • April 14, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • Parts: J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002
  • Gel Extraction
  • Parts: J23108_J61002, J23113_J61002
  • Eluted 50 µL
  • Lane # Contents Volume(µL)
    1 1 µL Dye, 5µL DNA kB ladder 6
    2 J23100_J61002 25
    3 J23100_J61002 25
    4 J23100_J61002 ~10
    5 J23108_J61002 25
    6 J23108_J61002 25
    7 J23108_J61002 ~10
    8 J23113_J61002 25
    9 J23113_J61002 25
    10 J23113_J61002 ~10
    11 J23114_J61002 25
    12 J23114_J61002 25
    13 J23114_J61002 ~10

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Restriction/Digestion

  • Master Mix: Yoyo Yao promoters x9
  • 35uL DNA, 14µL MM, 0.5µL Spe1, 0.5µL Pst1
  • Date: April 15, 2013
  • Master Mix Volume (μL)
    MIlliQ 36
    BSA 45
    10x buffer 2 NEB 45

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Restriction/Digestion

  • Master Mix: FS, KT ½ promoter ½ insulin/glucose x17
  • (Promoters) 25µL DNA, 24µL MM, 0.5µL Spe1, 0.5µL Pst1
  • (Insulin/Glucose) 25µL DNA, 24µL MM, 0.5µL EcoR1, 0.5µL Pst1
  • Date: April 15, 2013
  • Master Mix Volume (μL)
    MIlliQ 238
    BSA 85
    10x buffer 2 NEB 85

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1% Agarose Gel of Promoters

  • Names: Yoyo Y, Erin K
  • April 15, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • Lane # Contents Volume (μL)
    1 1 KB Ladder 6
    2 J23100-1 YYSP 25
    3 J23100-1 YYSP 25
    4 J23100-1 YYSP <10
    5 J23100-2 YYSP 25
    6 J23100-2 YYSP 25
    7 J23100-2 YYSP <10
    8 J23108-1 YYSP 25
    9 J23108-1 YYSP 25
    10 J23108-1 YYSP <10
    11 J23108-2 YYSP 25
    12 J23108-2 YYSP ~25
  • Lane # Contents Volume (μL)
    1 1 KB Ladder 6
    2 J23113-1 YYSP 25
    3 J23113-1 YYSP 25
    4 J23113-2 YYSP 25
    5 J23113-2 YYSP 25
    6 J23113-2 YYSP <10
    7 J23114-1 YYSP 25
    8 J23114-1 YYSP 25
    9 J23114-2 YYSP 25
    10 J23114-2 YYSP 25
  • Lane # Contents Volume (μL)
    1 1 KB Ladder 6
    2 J23100-1 YYSP 25
    3 J23100-1 YYSP 25
    4 J23100-1 YYSP 25
    5 J23100-2 YYSP 25
    6 J23100-2 YYSP <25
    7 J23108-1 YYSP 25
    8 J23108-1 YYSP 25
    9 J23108-1 YYSP 10
    10 J23108-2 YYSP 25
    11 J23108-2 YYSP 25
  • Lane # Contents Volume (μL)
    1 1 KB Ladder 6
    2 J23113-1 YYSP 25
    3 J23113-1 YYSP 25
    4 J23113-2 YYSP 25
    5 J23113-2 YYSP 25
    6 J23114-1 YYSP <10
    7 J23114-1 YYSP 25
    8 J23224-1 YYSP 25
    9 J23114-2 YYSP 10
    10 J23114-2 YYSP 25

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Transformation

  • Name: Dylan S
  • Date: April 17, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • Tube # Part # Plasmid Volume of Competent Cells(µL)
    1 J23100+J06702 J61002 100
    2 J23108+J06702 J61002 100
    3 J23113+J06702 J61002 50
    4 J23114+J06702 J61002 50
    5 J23100 mlc J06702 pSB3C5 50
    6 J23100 mlc J06702 pSB3C5 50
    +/- Controls PUC19 Amp resistance 50 each

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1% Agarose Gel of Promoter Constructs

  • Name: Chris I
  • Date: April 30, 2013
  • Species: E. coli
  • Strain: DH5α
  • Protocol: Ute Kothe
  • Lane # Contents Volume(µL)
    1 1 KB Ladder 6
    2 J23100_J61002 25
    3 J23100_J61002 25
    4 J23100_J61002 25
    5 J23100_J61002 25
    6 J23108_J61002 25
    7 J23108_J61002 25
    8 J23108_J61002 25
    9 J23108_J61002 25
    10 J23108_J61002 <20
    11 J23108_J61002 25
    13 1 KB Ladder 25
    12 J23100_J61002 <20
    1 1 KB Ladder 6
    2 J23113_J61002 25
    3 J23113_J61002 25
    4 J23113_J61002 <20
    5 J6702_pSB1AK2 20
    6 J6702_pSB1AK2 20
    7 J6702_pSB1AK2 20
    8 J6702_pSB1AK2 25
    9 J6702_pSB1AK2 25
    10 J6702_pSB1AK2 20
    11 J6702_pSB1AK2 20
    12 J6702_pSB1AK2 20
    13 None N/A

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