Team:Stony Brook/Notebook/Cancer-W7

I left you some of the commonly used templates down there under 8/8. Just fill them in as needed. I'll only really have wifi for a few hours on two days between Saturday and Thursday. If you need anything, message me on Facebook and I'll get back to you as soon as I can.

With the warmest love and regards,

~JOSHUA

Week 7: 8/8 - 8/14

PCR Table Template:
    Contents ul
    Phire #
    H2O #
    50 uM F Primer #
    50 uM R Primer #
    #ug/ul Construct #

PCR Settings Template:

    Phase Temperature (°C) Time (sec)
    Initial Denaturation 98 #
    Denaturation 98 #
    Annealing # #
    Extension 72 #
    Final Extension 72 #

Nanodrop Template:

    Content Concentration (ng/ul) 260/280
    Construct # #
    Vector # #

Ligation Template

    Content ul
    10X T4 Buffer #
    Vector # (# ng)
    Construct # (# ng)
    H2O #
    Ligase #

Multiple Digests Template:

This
    Content Experimental ul Control ul
    H2O # #
    Cutsmart # #
    EcoRI-HF # X
    XbaI # X
    DNA # #
Is
    Content Experimental ul Control ul
    H2O # #
    Cutsmart # #
    EcoRI-HF # X
    XbaI # X
    DNA # #

A
    Content Experimental ul Control ul
    H2O # #
    Cutsmart # #
    EcoRI-HF # X
    XbaI # X
    DNA # #
Thing
    Content Experimental ul Control ul
    H2O # #
    Cutsmart # #
    EcoRI-HF # X
    XbaI # X
    DNA # #


8/8

Sequencing

    T1 T2
    5.34 uL H2O 5.34 uL H2O
    1 uL CR-1 Primer 1 1 uL CR-1 Primer 2
    1.66 uL DNA (301.6 ng/uL) 1.66 uL DNA (301.6 ng/uL)

Diluted BioBrick MiniPreps for PCR

    BioBrick Name Concentration (ng/uL) Contents (DNA + water)
    K592009 76.9 1 uL + 6.69 uL water
    B0015 51.7 1 uL + 4.17 uL water
    K1033910 156.5 1 uL + 14.65 uL water
    R0082 49.5 1 uL + 3.95 uL water
    C0012 150.8 1 uL + 14.08 uL water
    J61100 123.0 1 uL + 11.3 uL water
    R0011 56.7 1 uL + 4.67 uL water

PCR of each BioBrick parts (25 uL Q5 MasterMix rxns)

    Contents uL
    10 uM VF2 primer 1.25 uL
    10 uM VR primer 1.25 uL
    DNA 0.5 uL
    Q5 MM 12.5 uL
    Water 19.5 uL


8/9

Gel run:
Lanes:
  1. Ladder
  2. R0011
  3. B001
  4. K1033
  5. R008
  6. J61100
  7. C0012
  8. K592
  • Didn't work

Nanodrop of Miniprepped Plamids:

    Tube # Concentration (ng/ul)
    1 277.3
    2 #
    2 #
    2 #

PCR of Minprepped Plasmids
    Contents ul
    Q5 Buffer 5uL
    H2O 16.25 uL
    10 uM F Primer 1.25 uL
    10 uM R Primer 1.25 uL
    Construct 0.5 uL
    Q5 Polymerase 0.25 uL
    Q5 Polymerase 0.25 uL

PCR Settings:

    Phase Temperature (°C) Time (sec)
    Initial Denaturation 98 30s
    Denaturation 98 20s
    Annealing 56 15s
    Extension 72 30s
    Final Extension 72 60s


8/10


25 ul PCR of Miniprepped Plasmids for Project

    Contents ul
    Q5 Buffer 5
    Q5 Polymerase 5
    H2O 16.25
    10 uM F Primer 1.25
    10 uM R Primer 1.25
    Construct 0.5
    Phase Temperature (°C) Time (sec)
    Initial Denaturation 98 30
    Denaturation 98 20
    Annealing 56 15
    Extension 72 30
    Final Extension 72 60
PCR was run on the following construct concentrations in ng/ul
  • 273
  • 176.5
  • 296.6
  • 311.5
  • 301.6
  • 301.6
  • 259
  • 366.6
  • PCRs did not work → Changed annealing temperature to 66°C
  • Re-ran the first 5 constructs on the above list

25 ul PCR of Miniprepped Plasmid for BioBrick Portion

    Contents ul
    Q5 Master Mix 12.5
    H2O 9.5
    10 uM F Primer 1.25
    10 uM R Primer 1.25
    Template 0.5
  • This setup was used for all BioBrick Parts
  • Ran with the "Sunil" Program with a 66°C annealing temperature


    • 8/11

    Ran a gel of BioBrick Plasmid PCRs from last night

    Lanes:
    1. BOOi5
    2. ROO82
    3. ROO11
    4. Ja1100
    5. Ladder
    6. K1033910
    7. K592009
    8. COO12
    • Used 8ul of each product was used in each gel

    25ul PCR Run

      Contents ul
      Q5 Buffer 5
      Q5 Polymerase 0.25
      H2O 15.75
      10 uM F Primer 1.25
      10 uM R Primer 1.25
      366.6ug/ul Construct 1ul
    PCR Settings - 35 Cycles
      Phase Temperature (°C) Time (sec)
      Initial Denaturation 98 30
      Denaturation 98 20
      Annealing 65 30
      Extension 72 30
      Final Extension 72 60
    Dilution of 366.6 Plasmid made

    (1ul)(366.6ng/ul) = X(1n/ul)


    Gel of Plasmid PCR

    Lanes
    1. Ladder
    2. Jonathan Plasmid
    3. Ryan Plasmid - 366.6 dilution
    Ran a PCR purification
    • Used the setup from 8/10
    • Also purified the BioBrick PCRs from 8/10

    Nanodropped the products

      Content Concentration (ng/ul) 260/280
      BOO15 15.1 1.72
      ROO15.1 15.1 1.85
      ROOd1 11.8 1.71
      J61100 11.3 1.61
      K59200 20.3 1.76
      K103340 21.7 1.82
      COO12 23.4 1.9

    Gel of BioBrick PCR

    Lanes:
    1. COO12
    2. ROO11
    3. K1033910
    4. J61100
    5. K592009
    6. ROO82
    7. BOO15

    Digest of Project Plasmids

    277.3 ng/ul Template
      Content Experimental ul Control ul
      H2O 40.89 41.39
      3.1 Buffer 5 5
      NotI 0.5 X
      DNA 3.61 3.61
    176.5 ng/ul
      Content Experimental ul Control ul
      H2O 38.83 39.33
      3.1 5 5
      NotI 0.5 X
      DNA 5.67 5.67
    • Band expected at 6500bp
    296.6 ng/ul
      Content Experimental ul Control ul
      H2O 41.13 41.63
      3.1 5 5
      EcoRI-HF 0.5 X
      DNA 3.37 3.37
    311.5 ng/ul
      Content Experimental ul Control ul
      H2O 41.29 41.29
      3.1 5 5
      EcoRI-HF 0.5 X
      DNA 3.21 3.21
    • Band expected at 300bp and 6200
    • Result = 1 band at 5600


    8/12

    Gels of project plasmid

    • Loaded with 20ul of each product in wells
    Gel 1
    Lanes:
    1. 277.3 Control
    2. 277.3 Experimental
    3. Ladder
    4. 176.5 Experimental
    5. 176.5 Control
    Gel 1
    Lanes:
    1. Empty
    2. 296.6 Control
    3. 296.6 Experimental
    4. Ladder
    5. 311.5 Experimental
    6. 311.5 Control


    Digested and nanodropped BioBrick parts

    Nanodropped the products

      Content Concentration (ng/ul) 260/280
      BOO15 11.9 1.39
      ROO11 10.2 1.38
      ROO82 18.4 1.64
      J61100 14.2 1.35
      K59200 9.5 1.46
      K103340 13.4 1.47
      COO12 13.6 1.47

    Gel of PEP352GAP + Jonathan Plasmids

    Lanes:
    1. 547
    2. 498.9
    3. Ladder
    4. Balazsi
    5. Transformed Balazsi

    Digests of BioBrick Parts - Round 1

    ROO11
      Content ul
      Cutsmart 5.5
      SpeI 0.5
      DNA 49
    J61100
      Content ul
      Cutsmart 3.98
      XbaI 0.5
      DNA 35.2

    K1033910
      Content ul
      Cutsmart 3.98
      SpeI 0.5
      DNA 37.3
    BOO15
      Content ul
      Cutsmart 4.75
      XbaI 0.5
      DNA 42.0

    ROO82
      Content ul
      Cutsmart 3.1
      SpeI 0.5
      DNA 27.2
    561100
      Content ul
      Cutsmart 3.98
      XbaI 0.5
      DNA 35.2

    K592009
      Content ul
      Cutsmart 5.9
      SpeI 0.5
      DNA 52.6
    561100
      Content ul
      Cutsmart 3.98
      XbaI 0.5
      DNA 35.2

    COO12
      Content ul
      Cutsmart 4.3
      SpeI 0.5
      DNA 38.5
    BOO15
      Content ul
      Cutsmart 4.75
      XbaI 0.5
      DNA 42.0

    PCR of IDT Construct

      Contents ul
      Phire 25
      H2O 22
      50 uM F Primer 1
      50 uM R Primer 1
      DNA Template 1
      Phase Temperature (°C) Time (sec)
      Initial Denaturation 98 30
      Denaturation 98 20
      Annealing 5 30
      Extension 72 30
      Final Extension 72 60

    Ran a gel on those products

    Lanes:
    1. Ladder
    2. Blank
    3. JK Construct Plasmid
    4. PCR

    Nanodrop of PCR

      Content Concentration (ng/ul) 260/280
      Construct 79.7 1.77


    8/13

    Miniprep and gel run of Dean vector

    • Two preps used Epoch kit
    • Two preps used Qiagen
    Ran the plasmids on a gel
    Lanes:
    1. 1st Epoch
    2. 2nd Epoch
    3. Ladder
    4. 1st Qiagen
    5. 2nd Qiagen
    • The gel wells 4 and 5 were contaminated by a little bit of RNA

    Nanodrop of Miniprepped plasmids

      Content Concentration (ng/ul) 260/280
      1st Epoch 133.4 1.89
      2nd Epoch 142.6 1.92
      1st Qiagen 188.5 2.02
      2nd Qiagen 205.6 1.97

    50ul Digest of Plasmid and Insert

    Epoch 1
      Content Experimental ul Control ul
      H2O 36.5 37.5
      Cutsmart 5 5
      PvuII-HF 0.5 X
      XhoI 0.5 X
      DNA 7.5 7.5
    Epoch 2
      Content Experimental ul Control ul
      H2O 37 38
      Cutsmart 5 5
      PvuII-HF 0.5 X
      XhoI 0.5 X
      DNA 7.01 7.01


    Insert
      Content Experimental ul Control ul
      H2O 31.5 32.5
      Cutsmart 5 5
      PvuII-HF 0.5 X
      XhoI 0.5 X
      DNA 12.55 12.55


    8/14

    Purified 8/13 digests with Qiagen PCR purificiation kit and ran a gel

    Lanes:
    1. Epoch 1 Control
    2. Epoch 1 Experimental
    3. Ladder
    4. Epoch 2 Control
    5. Epoch 2 Experimental
    6. Insert Experimental
    7. Insert Control

    Nanodropped digest purifications

      Content Concentration (ng/ul) 260/280
      Epoch 1 9.8 2.81
      Epoch 2 9.4 2.28
      Insert 15.8 1.98

    Troubleshooting

    • To fix digests, sequential diests attempted. Digested with only XhoI and heat deactivated. Then digested with PvuII. Purified it and nanodropped it
    Epoch 1
      Content Experimental ul Control ul
      H2O 37 37.5
      Cutsmart 5 5
      XhoI 0.5 X
      DNA 7.5 7.5
    Epoch 2
      Content Experimental ul Control ul
      H2O 37.5 38
      Cutsmart 5 5
      XhoI 0.5 X
      DNA 7.01 7.01
    • Placed in incubator
      • XhoII heat inactivated at 65°C for 20 minutes