Team:Stony Brook/Notebook/Cancer-W5

Week 5: 7/25-7/31

Week 5: 7/25-7/31




7/25

3X Phire Reactions (50ul)

    Contents ul
    Phire 25
    H2O 23.2
    50 uM F Primer 0.5
    50 uM R Primer 0.5
    95.1ug/ul Construct 0.8
  • PCR run with 30 cycles and annealing temperature at 66°C

Run on a gel

  • 10ul ladder
  • 5ul each PCR reaction
    • Ladder worked but lanes were smeared
    • Construct was left out over the weekend, so it was probably degraded

Three PCR reactions

  • Ran three PCRs with similar amounts of reagents as above, except each used a different concentration of template

Outreach

  • Team members presented iGEM information and projects and invited people to our Building With Biology event at the Seawolves Community Movie Night



7/26

Ran a gel of the two reactions from 7/25

  • Gel came out poorly

New 3 replicants of 50 ul PCR with Phire of IDT Construct

    Contents ul
    Phire 25
    F Primer 1
    R Primer 1
    Template 1/0.75/0.5
    H2O 22/22.25/22.25
  • PCR was run with 30 cycles at normal settings with the annealing temperature at 66°C

Ran a gel on the 3x Phire PCR products

  • Gel ended up looking good
  • Nanodropped it but didn't get a good concentration

Ran PCR on 28.9 ng/ul and 10.98 ng/ul constructs (20ul reactions)

Ran nanodrops on the following

    Content ng/ul 260/280
    Re-do of 28.9 ng/ul 29.1 1.9
    Re-do of 10.8 ng/ul 10.8 Bad
    7/26 Replicate of 10 ng/ul precursor 10.7 2.5
    7/26 of 10 ng/ul child 16.8 2.06

Experiment

  • Tried making another 10ng/ul child with longer PCR tubes
  • Nanodropped to 30.1 ng/ul




7/27

Ran a PCR on 8 reactions

  • TBA

Ran a gel on the 8 PCR products

Lanes:

  1. Ladder
  2. PCR 1
  3. PCR 2
  4. PCR 3
  5. PCR 4
  6. PCR 5
  7. PCR 6
  8. PCR 7
  • Ladder looked good but the PCR Products smeared

Ran a new gel including the digests + PCR product

Lanes:

  1. 10 ul Ladder
  2. PEP352GAP Experiment
  3. Empty
  4. PEP352GAP Control
  5. Construct Experiment
  6. Construct Control
  7. Empty
  8. PCR 8

Ran a PCR (50ul)

    Content ul
    Phire 25
    100 uM Fwd Primer 0.25
    100 uM Rev Primer 0.25
    10.8 ng/ul Template 1
    H2O 23.5

PCR Settings at 35 cycles:

    Phase Temperature (°C) Time (sec)
    Initial Denaturation 98 60
    Denaturation 98 25
    Annealing 66 20
    Extension 72 45
    Final Extension 72 60
  • Labelled "7/27 last hope CR-1 PCR"
  • Gel looked good
  • PCR purified + nanodropped → 14.6 ng/ul

Outreach

  • Cancer team and one member of the vaccine team went to present about iGEM, synthetic biology and our projects at the DNA Learning Labs at Cold Spring Harbor



7/28

PCR of 10.8 ng/ul Construct Sample

    Content ul
    Phire 25
    50uM F 0.5
    50 uM R 0.5
    10.8 un/ul Template 2
    H2O 22

PCR Settings: 35 Cycles

    Phase Temperature (°C) Time (sec)
    Initial Denaturation 98 30
    Denaturation 98 20
    Annealing 66 10
    Extension 72 30
    Final Extension 72 60

Digest: Mimicked Protocol from 7/18

  • PPEP352GAP : 498.4 ng/ul
  • Construct - 28.9 ng/ul
50 ul Reaction
    Content Experimental (ul) Control (ul)
    Water 42 43
    10x Cutsmart 5 5
    XnoI .5 X
    PvoII-HF .5 X
    498.4 ng/ul DNA 2 2
25 ul Reaction
    Content Experimental (ul) Control (ul)
    Water 4.2 5.2
    10x Cutsmart 2.5 2.
    XnoI .5 X
    PvoII-HF .5 X
    498.4 ng/ul DNA 17.3 17.3
  • Mixed reactions by flicking tube
  • Spin down in touches to mix
  • Incubate in incubator 37°C for an hour

Running gel of PCR Reaction + Digest

Lanes:

  1. PCR Reaction
  2. Construct Control
  3. Construct Digest
  4. Ladder
  5. Vector Digest
  6. Vector Control
  • Purified digest using Epoch Kit

Nanodrop of Digests

    Content Concentration (ng/ul) 260/280
    Construct 14.6 1.85
    Vector 14.6 1.80

20 ul Ligation of our hopes and dreams

    Content ul
    10X T4 Buffer 2
    Vector 4 (58.4 ng)
    Construct 1.55 (22.69 ng)
    H2O 11.45
    Ligase 1
  • Gently Mixed Reaction
  • Incubate at room temperature 2 hours
  • Chill on ice and put in freezer

Transformations

  • Transformed Emmanuel + Ryan's ligations, control + piggybac's
  • 1:10 dilution made of each transformation
  • Created LB Cultures
  • Incubated
  • PUC19 Control grown on chloramp plates
  • Did not survive



7/29

  • LB Cultures (8ml each) setpup for Ryan's Ligation, Emmanuel's Ligation, PiggyBac



7/30

Miniprep of Ligated + Transformed vector cultures

  • Spun at 4000rpm for 5 minutes at 23°C
  • Spun an additional 2 minutes at 4000rpm

Mixed Epoch McI Buffer into Qiagen Protocol

  • 250ul of MXI used for 8ml LB Preps
  • Lysis Rxn Sitting fo 4 minutes
  • Eluted with 2 step of 2ul nuclease free NEB Water

Nanodropped miniprepped plasmids - Water as background

    Content Concentration (ng/ul) 260/280 Graph Character
    PiggyBac 121.2 1.91 Lit
    PiggyBac 1:10 423.7 1.87 Lit
    Emmanuel 193 1.92 Lit
    Ryan 446.4 1.89 Lit
    Ryan 1 226.3 1.94 Lit
    Ryan 2 232.2 1.93 Lit
    Ryan 3 272.6 1.90 Lit
    Ryan 4 335.5 1.84 Lit

Running gels with miniprepped plasmids from earlier today

Lanes:

  1. PiggyBac: 5ul + 1 ul dye
  2. PiggyBac: 2.5 ul + 1 dye
  3. Ladder
  4. Ryan: 2.5 ul + 1 ul dye
  5. Ryan 1: 4.17ul + .83 ul dye
  6. Ryan 2: 4.17ul + .83 ul dye
  7. Ryan 3: 4.17ul + .83 ul dye
  8. Ryan 4: 4.17ul + .83 ul dye
  • Ran at 116V for 65 minutes
  • Gel looked strange

Prepped digest of Lanes 1, 2, 4 and 7
  • XbaI, EcoRI, XhoI and PvoII-HF respectively
Lane 1: PIggyBac - 121.2 ng/ul
    Content Experimental ul Control ul
    H2O 35.75 36.75
    Cutsmart 5 5
    EcoRI-HF 0.5 X
    XbaI 0.5 X
    DNA 8.25 8.25
Lane 2: PIggyBac 1:10 - 423.7 ng/ul
    Content Experimental ul Control ul
    H2O 41,6 42.6
    Cutsmart 5 5
    EcoRI-HF 0.5 X
    XbaI 0.5 X
    DNA 2.36 2.36

Lane 4: Ryan - 446.4 ng/ul
    Content Experimental ul Control ul
    H2O 41.8 42.8
    Cutsmart 5 5
    EcoRI-HF 0.5 X
    XbaI 0.5 X
    DNA 2.24 2.24
Lane 7: Ryan 3 - 272.6 ng/ul
    Content Experimental ul Control ul
    H2O 40.3 41.3
    Cutsmart 5 5
    EcoRI-HF 0.5 X
    XbaI 0.5 X
    DNA 3.67 3.67

Ran gel of digest

  • Added 10ul of dye to each tube to stop reaction
  • Ran at 116V for 35 minutes
Gel #1

Lanes:

  1. PiggyBac Control
  2. PiggyBac Experimental
  3. Ladder
  4. PiggyBac 1:10 Experimental
  5. PiggyBac 1:10 Control
Gel #2

Lanes:

  1. Ryan Control
  2. Ryan Experimental
  3. Ladder
  4. Ryan 3 Experimental
  5. Ryan 3 Control



7/31

Digest of Ryan and PB 1:10

  • Redid the digest
  • Miniprepped with phosphatase treatment for the digest
  • Used 5 units/ul of Antarctic Phosphatase
Lane 2: PIggyBac 1:10 - 423.7 ng/ul
    Content Experimental ul Control ul
    H2O 41,6 42.6
    Cutsmart 5 5
    EcoRI-HF 0.5 X
    XbaI 0.5 X
    DNA 2.36 2.36
Lane 4: Ryan - 446.4 ng/ul
    Content Experimental ul Control ul
    H2O 41.8 42.8
    Cutsmart 5 5
    EcoRI-HF 0.5 X
    XbaI 0.5 X
    DNA 2.24 2.24

Post digest procedures

  • Flicked tubes and spun down in touches
  • Incubated at 37°C for an hour
  • Purified with Epoch kit
  • Phosphatase and heat inactivated at 80°C for 2 minutes
  • 4ul of phosphate buffer, 1 ul of phosphatase and 5ul of nuclease free H2O used

Ran a gel

  • Used 8ul loading dye in each tube

Lanes:

  1. PB Control
  2. PB Experimental
  3. Ladder
  4. Ryan Experimental
  5. Ryan Control

Ran a digest of Dean vector with various enzymes control

  • 25ul reaction - 498.ng/ul
Xhol
    Content l ul
    H2O 21
    Cutsmart 2.5
    XhoI 0.5
    DNA 1
PvoII-HF
    Content ul
    H2O 21
    Cutsmart 2.5
    PvoII 0.5
    DNA 1

No Enzyme - Control
    Content ul
    H2O 21.5
    Cutsmart 2.5
    DNA 1
Both Enzymes
    Content Experimental ul
    H2O 20.5
    Cutsmart 2.5
    XhoI 0.5
    PvoII-HF 0.5
    DNA 1

Running a gel

Lanes:

  1. XhoI
  2. PvoII-HF
  3. Ladder
  4. Both Enzymes
  5. No enzyme

New 25ul Digest of Ryan and PB 1:10 miniprep

PB1:10 (423.7ng/ul)
    Content Experimental (ul) Control (ul)
    H2O 20.3 21.3
    Cutsmart 2.5 2.5
    XbaI 0.5 X
    EcoRI-HF 0.5 X
    DNA 1.18 1.18
Ryan (446.4 ng/ul)
    Content Experimental (ul) Control (ul)
    H2O 20.4 21.4
    Cutsmart 2.5 2.5
    XbaI 0.5 X
    EcoRI-HF 0.5 X
    DNA 1.12 1.12

Gel run with 10ul of each

Lanes:

  1. PB Control
  2. PB Experimental
  3. Ladder
  4. Ryan Experimental
  5. Ryan Control