Team:Stony Brook/Notebook/Cancer-W9

Week 9: 8/22 - 8/28

Week 9: 8/22 - 8/28



8/22

PCR of new IDT Shipments

AmilCP
    Contents ul
    Q5 MasterMix 12.5
    H2O 9
    10 uM F Primer 1.25
    10 uM R Primer 1.25
    Template 1
IDT #1
    Contents ul
    Q5 MasterMix 12.5
    H2O 6.5
    10 uM F Primer 2.5
    10 uM R Primer 2.5
    Template 1

IDT #2
    Contents ul
    Q5 MasterMix 12.5
    H2O 6.5
    10 uM F Primer 2.5
    10 uM R Primer 2.5
    Template 1

PCR Settings: 25 Cycles

    Phase Temperature (°C) Time (sec)
    Initial Denaturation 98 30
    Denaturation 98 10
    Annealing 65 30
    Extension 72 120
    Final Extension 72 120

Digest of BioBricks


J23119 - 225.8ng
Content Experimental ul Control ul
Cutsmart 5 5
EcoRI-HF 0.5 X
SpeI 0.5 X
DNA 44.3 2.2
B0015 - 201.5ng
Content Experimental ul Control ul
Cutsmart 5 5
XbaI 0.5 X
PstI 0.5 X
DNA 49.6 2.5
E0040 - 179.3ng
Content Experimental ul Control ul
Cutsmart 5 5
EcoRI-HF 0.5 X
SpeI 0.5 X
DNA 55.8 2.8

LB Prep

J23119
  • 8ml LB
  • 2ml chloramphenicol
K592009
  • 8ml LB
  • 2ml chloramphenicol
E0040
  • 8ml LB
  • 2ml Ampicillin
B0015
  • 8ml LB
  • 2ml chloramphenicol


8/23

PCR of amilCP, #1 and #2 with 67° annealing temperature

  • Ran a gel afterwards

Lanes:
  1. Ladder
  2. AmilCP
  3. #1
  4. #2

Gel of 10ug overnight BB digest

Lanes:
  1. Ladder
  2. J23119
  3. J23119 Control
  4. B0015
  5. B0015 Control
  6. E0040
  7. E0040 Control

Competent Cells (TSS)

50ml TSS
Content Amount
PEG3350 5g
1M MgCL2 1.5 ml
DMSO 2.5ml
LB Fill to 50ml
  • Tested pH to be ~6.5

New PCR of Toggle #1 and amil CP

  • 69°C annealing temperature
  • Extension length - 2.5 mins
  • Ran for 30 Cycles

Ran a gel

Lanes:
  1. amil CP
  2. #4
  3. Ladder

Gel of Digested BB Parts

Lanes:
  1. Ladder
  2. J23119 #1
  3. J23119 #2
  4. B0015 #1
  5. B0015 #2
  6. E0040 #1
  7. E0040 #2

Nanodrops of BioBricks and PCR purifications

Content Concentration (ng/ul) 260/280
R592009 234.3 1.92
J23119 93.0 1.95
B0015 229.4 2.00
E0040 287.5 1.95
PiggyBac 274.0 1.89
Toggle #2 9.6 2.45

Digests

  • Re-do of the the digest from 8/21

PCR of Amil CP and #1

  • 67°C annealing temperature for 15 seconds
  • 60 second extension

25ul PCR of Toggle #2

  • 68°C annealing temperature
  • Primer concentrations at 100-500 ng/ul

Gel of BioBrick Digests

Gel #1
Lanes:
  1. Ladder
  2. B0015
  3. B0015
  4. B0015 Control
  5. J12119
  6. J23119
  7. J23119 Control
Gel #2
Lanes:
  1. Ladder
  2. K592009
  3. K592009
  4. K592009
  5. K592009 Control
  6. E0040
  7. E0040
  8. E0040 Control

PCR of Toggle #2 - 30x

Contents ul
Q5 MasterMix 12.5
H2O 5.5
10 uM F Primer 1.0
10 uM R Primer 1.0
Template 5
Contents ul
Q5 MasterMix 12.5
H2O 3.5
10 uM F Primer 2.0
10 uM R Primer 2.0
Template 5

Gel

Lanes:
  1. Ladder
  2. Toggle #2 - 1ul primer
  3. Toggle #2 - 2ul primer

Toggle #1 Trials

PCR of Toggle #1

  • Ran 6 trials. The first started with 0.5 ul of each of the F and R primers.
  • Each trial increased by 0.5 until we got to 2.5ul of primers
  • Ran a 0.25ul primer trial
  • Used 12.5ul of Q5 MasterMix and 5ul of template

Toggle #1 Gel

Lanes:
  1. Ladder
  2. 0.25
  3. 0.5
  4. 1.0
  5. 1.5
  6. 2.0
  7. 2.5


New PCR of Toggle #1 - Same settings as earlier Toggle #1

Contents ul
Q5 MasterMix 12.5
H2O X
5 uM F Primer 4
5 uM R Primer 4
Template 4.5
  • Also ran PCRs with 2.0 and 2.5 ul of primers


8/24



8/25

Nanodrop of PCR products

PCR Products
Content Concentration (ng/ul) 260/280
2.0 #1 43.7 1.87
2.5 #1 46.9 1.91
4.0 #1 54.7 1.94
1.0 #2 13.8 1.86
2.0 #2 31.1 2.18
BB Gel Extracts
Content Concentration (ng/ul) 260/280
J23119 3.8 2.68
E0040 13.2 2.19
B0015 8.7 1.45
K592009 41.3 1.89

PCR of Toggle #2

Contents ul
Q5 MasterMix 12.5
H2O 6.5
5 uM F Primer 0.5
5 uM R Primer 0.5
Template 5
  • Also ran PCRs with 1.5, 2.5 and 3.5 ul of primers


BioBrick Digests

K592009 - 234.3ng/ul
    Content Experimental ul 25 ul Control
    H2O X 20.37
    Cutsmart 15.8 2.5
    EcoRI-HF 1.0 X
    SpeI 1.0 X
    DNA 140 2.13
J23119 - 93.0ng/ul
    Content Experimental ul 25 ul Control
    H2O X 17.12
    Cutsmart 15.8 2.5
    EcoRI-HF 1.0 X
    SpeI 1.0 X
    DNA 140 5.38

B0015 - 229.4ng/ul
    Content Experimental ul 25 ul Control
    H2O X 20.32
    Cutsmart 15.8 2.5
    XbaI 1.0 X
    PstI 1.0 X
    DNA 140 2.13
E0040 - 287.5 ng/ul
    Content Experimental ul 25 ul Control
    H2O X 20.76
    Cutsmart 15.8 2.5
    EcoRI-HF 1.0 X
    SpeI 1.0 X
    DNA 140 1.74

PCR Purification and Nanodrop of Toggle 2

Tube # Concentration (ng/ul) 260/280
#2 12.3 1.36
#3 27.3 1.49

Digests of Toggles 1 and 2

Toggle 1
    Content Experimental ul
    H2O 12.36
    2.1 Buffer 2.5
    EcoRI-HF 0.5
    SpeI 0.5
    DNA 9.14
Toggle 2
    Content Experimental ul
    H2O 12.36
    2.1 Buffer 2.5
    EcoRI-HF 0.5
    SpeI 0.5
    DNA 9.14

Ligation

Content ul
2.1 Buffer 5
B5A 0.5
Backbone 4ul
EcoRI-HF 0.5
PstI 0.5
T4 Mastermix 4ul
H2O 18.5


8/26

Met with Dr. Zainul about our project

  • Test would have to be administered in a lab
  • Only on an at risk population (family history, heavy alcohol consumption, etc.)
  • Would need to get the protocol down and license to sell it to a lab
  • Not all private practices have centrifuges, so doing the test with a visually detectable indicator won't work
  • Not all private practices have UV lights, so the draw of this being fast may not be as great as we thought
  • Maybe consider light chromatography
  • If this can be done in a private practice, we'll need a sort of stick test, similar to the glucose and protein tests for urine
  • Can consider coupling GPC-1 binding to secretion of a selected product that can bind to an external indicator that can be poured into the solution, like phenolphthalein


8/28

PCR of new IDT Shipments

AmilCP
    Contents ul
    Q5 MasterMix 12.5
    H2O 7
    5 uM F Primer 0.25
    5 uM R Primer 0.25
    100 ug/uL Template 5
  • The 5 uM F primers and the 5 uM R primers went up in increments of 0.5 uL until we reached 2.5 uL
  • The concentration of water started at 7 uL and went down by 0.5 uL for each successive PCR until we reached 2.5 uL

PCR Settings Template:

    Phase Temperature (°C) Time (sec)
    Initial Denaturation 98 30s
    Denaturation 98 10s
    Annealing 67 15s
    Extension 72 60s
    Final Extension 72 120s