Team:Stony Brook/Notebook/Cancer-W8

Week 8: 8/15 - 8/21

Week 8: 8/15 - 8/21



8/15

Gel of purified digests from 8/14

Lanes:

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

PCR purified and nanodropped the digests

    Content Concentration (ng/ul) 260/280
    Epoch 1 9.2 2.26
    Epoch 2 7.9 1.91

Ran another digest

Qiagen 1 (Both enzymes)
    Content Experimental ul Control ul
    H2O 18.85 19.85
    Cutsmart 2.5 2.5
    PvuII-HF 0.5 X
    XhoI 0.5 X
    DNA 2.65 2.65
Qiagen 1 (Sequential)
    Content Experimental ul Control ul
    H2O 19.85 19.85
    Cutsmart 2.5 2.5
    XhoI 0.5 X
    DNA 2.65 2.65

Qiagen 2 (Both enzymes)
    Content Experimental ul Control ul
    H2O 19.07 20.07
    Cutsmart 2.5 2.5
    PvuII-HF 0.5 X
    XhoI 0.5 X
    DNA 2.43 2.43
Qiagen 2 (Sequential)
    Content Experimental ul Control ul
    H2O 19.57 20.07
    Cutsmart 2.5 2.5
    Enzymes 0.5 X
    DNA 2.43 2.43

498.9 ng/ul Plasmid
Both Enzymes
    Content Experimental ul Control ul
    H2O 20.5 21.5
    Cutsmart 2.5 2.5
    PvuII 0.5 X
    XhoI 0.5 X
    DNA 1.0 1.0
Sequential
    Content Experimental ul Control ul
    H2O 21 22
    Cutsmart 2.5 2.5
    Enzymes 0.5 X
    DNA 1.0 1.0

Ran gels of plasmid digets

Gel 1

Lanes:

  1. Qiagen 1 Control
  2. Qiagen 1 (Both)
  3. Qiagen 1 (Sequential)
  4. Ladder
  5. Qiagen 2 (Sequential)
  6. Qiagen 2 (Both)
  7. Qiagen 2 Control
Gel 2

Lanes:

  1. Ladder
  2. Old Sequenced
  3. Old Both
  4. Old Control

Ran gel of plasmids for gel extract

Lanes:

  1. Ladder
  2. Qiagen 1 Sequential
  3. Qiagen 1 Both
  4. Quaigen 2 Sequential
  5. Qiagen 2 Both

Troubleshooting

Ran a gel and digest to diagnose if PvuII was denatured

205.6ng/ul

    Content Experimental ul Control ul
    H2O 19.6 20.1
    Cutsmart 2.5 2.5
    PvuII 0.5 X
    DNA 2.43 2.43

Gel of digests from the morning and PvuII-HF diagnostic

Lanes:
  1. Qiagen 1 Control
  2. Qiagen 1 Both
  3. Qiagen 1 Sequential
  4. Qiagen 2 Both
  5. Qiagen 2 Sequential
  6. Ladder
  7. PvuII Diagnostic
  8. PvuII Diagnostic Control
  • Plasmid extracted from gel

Nanodrop of plasmid gel extract

    Content Concentration (ng/ul) 260/280
    Plasmid Gel Extract 7.1 1.45


8/16


Diagnostic PvuII-HF Digest

188.1 ng/ul
Content Experimental ul Control ul
H2O 4.25 4.85
Cutsmart 2.5 2.5
PvuII-HF 0.5 X
DNA 2.65 2.65
205.6 ng/ul
Content Experimental ul Control ul
H2O 4.17 5.07
Cutsmart 2.5 2.5
PvuII-HF 0.5 X
DNA 2.43 2.43

Ran a gel on PvuII Digest

Lanes:
  1. Ladder
  2. 205.6 Control
  3. 205.6 Digest
  4. 188.5 Control
  5. 188.5 Digest

Nanodropped PvuII Digest

    Content Concentration (ng/ul) 260/280
    188.5 1.7 1.36
    205.6 1.4 2.05

PCR of BioBrick Parts



J23119
    Contents ul
    Q5 MasterMix 12.5
    H2O 6,5
    10 uM F Primer 2.5
    10 uM R Primer 2.5
    Template 1
E0040
    Contents ul
    Q5 MasterMix 12.5
    H2O 6,5
    10 uM F Primer 2.5
    10 uM R Primer 2.5
    Template 1
BOO15
    Contents ul
    Q5 MasterMix 12.5
    H2O 6,5
    10 uM F Primer 2.5
    10 uM R Primer 2.5
    Template 1


8/17

Gel of old PEP352GAP plasmids to test viability + Gel of BioBrick PCRs

Gel #1
Lanes:
  1. 498.4
  2. 311.5
  3. 259.0
  4. 296.6
  5. Ladder
  6. 176.5
  7. 277.3
  8. 366.6
Gel #1
Lanes:
  1. 133.4
  2. 188.5 PvuII
  3. 205.6 PvuII
  4. 142.6
  5. Ladder
  6. J23119
  7. E0040
  8. B0015
  • 142.6 was thrown out
  • Gel #1 looked a lot better than #2


100ul Sequential digest - PvuII and then XhoI

498.4 ng/ul
Content Experimental (100) ul Control (25) ul
H2O 85 21.5
Cutsmart 10 2.5
PvuII-HF 1 X
DNA 4.00 1.00
366.6 ng/ul
Content Experimental (100) ul Control (25) ul
H2O 83.5 21.14
Cutsmart 10 2.5
PvuII-HF 1 X
DNA 5.46 1.36
  • Incubated at 37°C for an hour

Nanodropped PvuII Digest

    Content Concentration (ng/ul)
    498.4 14.2
    366.6 29.6

XhoI Digest

29.6
Content Experimental ul Control ul
H2O 2.6 3.1
Cutsmart 5.0 5.0
XhoI 0.5 X
DNA 16.9 16.9
  • Incubated at 37°C for an hour


Ran a gel on sequential digests

Lanes:
  1. Ladder
  2. 29.6
  3. 29.6
  4. 14.2


Ligation of Dean Vector + Insert

    Content ul
    10X T4 Buffer 2.0
    Vector 3.0
    Insert 9.0
    H2O 5.0
    Ligase 1.0


8/18



8/19

Miniprep of Dean vector and BioBricks

  • Four tubes of Dean vector
  • Three tubes of BioBricks

    • Nanodrop of Miniprepped Dean Vectors + BioBricks

      Content Concentration (ng/ul) 260/280
      J23119 166.3 1.96
      B0015 137.4 1.93
      E0040 139.8 1.98
      DV #1 365.5 1.92
      DV #2 230.5 1.93
      DV #3 282.3 1.94
      DV #4 113.0 1.91

Initial Digests

J23119 - 166.3ng/ul
    Content Experimental ul Control ul
    H2O 38 39
    Cutsmart 5 5
    EcoRI-HF 0.5 X
    SpeI 0.5 X
    DNA 6 6
J61100 - 1.85ng/ul
    Content Experimental ul Control ul
    H2O 44 45
    Cutsmart 5 5
    XbaI 0.5 X
    PstI 0.5 X
    DNA 0.5 0.5

E0040 - 139.8/ul
    Content Experimental ul Control ul
    H2O 36.9 37.9
    Cutsmart 5 5
    EcoRI-HF 0.5 X
    SpeI 0.5 X
    DNA 7.15 7.15
B0015- 137.4ng/ul
    Content Experimental ul Control ul
    H2O 36.7 37.7
    Cutsmart 5 5
    XbaI 0.5 X
    PstI 0.5 X
    DNA 7.28 7.28

Gel of Digests

Gel #1
Lanes:
  1. J23119 C
  2. J23119 E
  3. J23119 E #2
  4. Ladder
  5. E0040 E
  6. E0040 E #2
  7. E0040 C
Gel #2
Lanes:
  1. J61100 C
  2. J61100 E
  3. J61100 E #2
  4. Ladder
  5. B0015 E
  6. B0015 E #2
  7. B0015 C


8/20



Digests of GFP, Promoter, and Terminator

Promoter
    Content Experimental ul Control ul
    H2O 32 33
    Cutsmart 5 5
    EcoRI 0.5 X
    SpeI 0.5 X
    DNA 12 12
GFP
    Content Experimental ul Control ul
    H2O 29.7 30.7
    Cutsmart 5 5
    EcoRI-HF 0.5 X
    SpeI 0.5 X
    DNA 14.3 14.3
Terminator
    Content Experimental ul Control ul
    H2O 29.4 30.4
    Cutsmart 5 5
    XbaI 0.5 X
    PstI 0.5 X
    DNA 14.6 14.6


8/21

Nanodrop of BB Component MiniPreps

    Content Concentration (ng/ul) 260/280
    B0015 201.5 1.99
    J23119 225.8 1.99
    E0040 179.3 2.03