Team:CU-Boulder/Notebook

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Notebook Timeline

Welcome to our notebook! As you scroll down the timeline you can see our actual notes for what we did each day.
2019
13 May

For the 2A9N - remove the H and the L chains from sequence (only want the M and I).

Need to do:

  • Make the 9 Bsa1 sites
  • Check NEB digest - get above 90% chance of working/not binding with the wrong overhang
  • Check methylation sites

14 May

Bsa1 cutting sites--9 regions and 9 cutting sites

Region 1 - linker region 1 (ScFv1 and AraC1)

  • Bsa1 in ScFv1 is TTAC
  • Bsa1 in AraC1 is AGTG
  • 321 bases

    • Region 2 - AraC1 and ribo linker and ScFv2

  • Bsa1 in AraC1 is AGTG
  • Bsa1 in ScFv2 is CAGA
  • 580 bases

    • Region 3 - Linker region 2 (ScFv2 and AraC2)

  • Bsa1 in ScFv2 is CAGA
  • Bsa1 in Arac2 is TGAA
  • 361 bases

    • Region 4 - AraC2 and part of plasmid (1)

  • Bsa1 in AraC2 is TGAA
  • Bsa1 in Plasmid (1) is TGCC
  • 306 bases
    • There is extra stuff added on. The Bsa1 overhang starts 14 bases in

    Region 5 - Plasmid (1) (Plasmid 1 w/ enzymes)

  • Bsa1 1 is TGCC
  • Bsa1 2 is AAAA
  • 638 bases

    • Region 6 - Plasmid (2) to Plasmid (3) (Plasmid 2 w/ enzymes)

  • Bsa1 1 is AAAA
  • Bsa1 2 is CCAG
  • 1449 bases

    • Region 7 - Plasmid (3) and Plasmid (4) (Plasmid 3 w/ enzymes)

  • Bsa1 1 is CCAG
  • Bsa1 2 is GCAA
  • 1296 bases

    • Region 8 - Plasmid (4) and Plasmid (4) with the ScFv1 (Plasmid 4 with enzymes)

  • Bsa1 1 is GCAA
  • Bsa1 2 is ATTC
  • 1694 bases

    • Region 9 - Plasmid (4) with the ScFv1

  • Bsa1 in Plasmid (4) is ATTC
  • Bsa1 in ScFv1 is TTAC
  • 405 bases
    • 48 bases from the plasmid are with the ScFv1 including the overhang

    15 May

    From Twist: 100 ng - 1 μg for each fragment

  • Concentration - in 2 ml tube with 10ng/μl

    • Fragments were ordered today.. expect to recieve them by June 12th. Next order by date is June 12 - get by July 10th

    23 May

    Chain M (Scfv2) in the structure of Fluro antibody missing residue 149 (Gly)

  • ScFv1 = Chain I = Chain A (ScFv1 + AraC1)
  • ScFv2 = Chain M = CHain B (ScFv2 + AraC2)

    • 3 June

    Continued to work on modeling and grants.

  • Linker addition onto Arac1 (Chain C) = 10 amino acids -> 1 GSGSGSGRGQ 10
  • Linker addition onto Arac2 (Chain D) = 15 amino acids -> 1 SGSGSGSGSGSGRGQ 15

    • A -> C = Scfv1 + Arac1. B -> D = Scfv2 + Arac2

    4 June

    Continued modeling and fixing different linkers

    5 June

    ***Linker additions made to DNA fragments on 6/3 changed scfv1=A, scfv2=C, arac1=B, arac2=D on pymol files

  • A -> B = Scfv1 + Arac1
  • C -> D = Scfv2 + Arac2

    • 6 June

    For control, IgG1 ~ 1ngq antibody - humanized

  • M -> L = scfv2 + fab2
  • I -> H = scfv1 + fab1

    • 10 June

    Received DNA from Twist, will bring up DNA to create stock solutions tomorrow.

    11 June

    TEST

    12 June

    DNA IN EACH TUBE:

  • 3.9 pmol = 3.9 e 10^-12 mol - plasmid4 + scfv1
  • 4.9 pmol = linker region 1
  • 2.75 pmol = arac1 + scfv2
  • 4.3 pmol = linker region 2
  • 4.9 pmol = acrc2 + plasmid 1
  • 2.5 pmol = plasmid 1
  • 1.11 pmol = plasmid 2
  • 1.2 pmol = plasmid 3
  • 0.92 pmol = plasmid 4

    • AMOUNT OF Tris TO ADD TO BRING UP TWIST DNA:

  • Add 97.5 μl to plasmid4 + scfv1
  • Add 122.5 μl to linker region 1
  • Add 68.75 μl to scfv1 + arac2
  • Add 107.5 μl to linker region 2
  • Add 122.5 μl to acrc2 + plasmid 1
  • Add 62.5 μl to plasmid 1
  • Add 27.75 μl to plasmid 2
  • Add 30 μl to plasmid 3
  • Add 23 μl to plasmid 4

    • Each piece of our DNA plasmid should now have a concentration 40 fmol/1μl

    ADDITION OF AMP TO AGAR MIX:

  • 100 mg/ml - 2ml per 1 L

    • 13 June

    TEST

    14 June

    TEST

    15 June

    TEST

    16 June

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    17 June

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    18 June

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    19 June

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    20 June

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    21 June

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    22 June

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    23 June

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    24 June

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    25 June

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    26 June

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    27 June

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    28 June

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    29 June

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    30 June

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    31 June

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    1 July

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    2 July

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    3 July

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    4 July

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    5 July

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    6 July

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    7 July

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    8 July

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    9 July

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    10 July

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    11 July

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    12 July

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    13 July

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    14 July

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    15 July

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    16 July

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    17 July

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    18 July

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    19 July

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    20 July

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    21 July

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    22 July

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    23 July

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    24 July

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    25 July

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    26 July

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    27 July

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    28 July

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    29 July

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    30 July

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    July

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    1 August

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    2 August

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    3 August

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    4 August

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    5 August

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    6 August

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    7 August

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    8 August

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    10 August

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    11 August

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    12 August

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    13 August

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    14 August

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    15 August

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    17 August

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    19 August

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    20 August

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    22 August

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    23 August

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    24 August

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    25 August

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    26 August

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    27 August

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    28 August

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    29 August

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    30 August

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