Team:Stony Brook/Notebook/Vaccine-W4

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Week 4: 7/18-

7/18

Ran a 50ul PCR
    Content Amount (ul)
    BB Prefix 2.5
    BB Suffix 2.5ul
    Q5 25
    IDT Original Construct 1ul
    H2O 19
Ran a second 50ul PCR
    Content Ax (ul) Ay (ul) A2 (ul)
    Phire 25 25 25
    BB Fwd 2.5 2.5 2.5
    BB Rev 2.5 2.5 2.5
    Template 4 2 4
    H2O 16 18 16
  • Initial denaturation was 2 minutes and 30 seconds
  • Let it heat before adding primers
Ran a third 50ul PCR
    Content BC (ul) BB (ul)
    Phire 25 25
    Gal Fwd .25 .25
    BB Rev .25 .25
    Template 1 2
    H2O 23.5 23.5
    • Ran a gel with Ladder 1 and Ax, Ay and Az, each at 2.5 ul
    • On the same gel, ran Ladder 2 with BB and BC, each at 5 ul

7/20

Ran a gel

Lane:

  1. 10ul ladder
  2. 2ul loading dye + 10ul Gal + Construct ligation (green tube)
  4. 2ul loading dye + 10ul Gal + Construct ligation (pink tube)
    Content BE (ul)
    Phire 25
    Gal Fwd .25
    BB Rev .25
    Template (ligation solution raw) 2
    Nuclease-free H2O 22.5
    PCR settings for 35 cycles:
      Phase Time (s) Temperature (°C)
      Initial Denaturation 60 98
      Denaturation 30 98
      Annealing 20 65
      Extension 30 72
      Final Extension 60 72
    • Held at 4°C

7/21

PCR Run - iGEM Vol1
    Contents Gal (ul) Construct (ul)
    Gal .5 X
    Construct X .5
    Gal F Primer .25 X
    Prefix-F X .25
    Suffix-R .25 .25
    H2O 24 24
    Phire 25 25
Ran a gel

Lane:

  1. Ladder
  2. Gal PCR
  3. Construct PCR
  • PCR purified using a NEB Monarch Kit

7/22

Nanodropped Gal and Construct
  • Gal → 141.6 ug/ul
  • Con → 83.9 ug/ul
Gal 10 ul Reaction
    Content ul
    Nuclease-free H2O 1.44
    10X Buffer 1ul
    HF-SpeI 0.5
    DNA 7.06
  • SpeI (To deactivate the restriction) at 80°C
Construct 50 ul Reaction
    Content ul
    Nuclease-free H2O 1.5
    10X Buffer 1ul
    XbaI 0.5
    DNA 11.92
  • Ran the XbaI (for ligation) at 65°C