Team:Stony Brook/Notebook/Cancer-W3

Week 3: 7/11-7/17

Week 3 (7/11-7/17)



7/11


  • Ran PCR on the construct (5x) → gel did not work
  • PCR'ed 84.2 ng/ml construct
  • 5 replicates made using Phire

Thermocycler Settings: 35 Cycles

    Step Temperature (Degrees C) Duration (seconds)
    Initial Denaturation 98 30
    Denaturation 98 20
    Annealing 66 5
    Extension 72 30
    Final Extension 72 60
    Hold 4 Hold

Ran a PCR

    Content ul
    Water 19.5 uL
    Phire Polymerase 25 uL
    Template 0.5 uL
    Forward Primer 2.5 uL
    Reverse Primer 2.5 uL
  • Restreaking of YEP352GAP transformed cells for miniprep
  • Restreaking placed in incubator at 12am
  • Made gel for electrophoresis of PCR construct → it didn't work


7/12

Ran a PCR: Same settings as 7/11

    Content ul
    Q5 Rx Buffer 10 uL
    10nM dNTPs 1 uL
    10 uM Forward Primer 2.5 uL
    10 uM Reverse Primer 2.5 uL
    Template (Q5 PCR Pw.1 for 6/30/16) 0.5 uL
    Q5 Polymerase 2.5 uL
    Water 33 uL
  • LB liquid culture (3ml with 3ul of ampicillin)
  • Loaded 10ul Ladder DNA.
  • Loaded 1ul dye + 5ul PCR product
  • Ran gel at 115V
  • Checked gel ladder → faint, no band for the PCR product
  • Made new gel, loaded with the same amount of reagents as before → ran at 90V → No ladder band
  • Ran it again at 115V → visible ladder, no PCR band
  • Preparing a gel to run ladder and construct that previously showed a strong band → used this to diagnose a problem with gel setup. Ran TDGF1 152.9 Phire Child. Lane 1 is ladder, Lane 2 is that construct (10ul) with 2ul of dye
  • Setup new PCR using the 6/30/16 PCR construct
  1. 98°C → 30sec
  2. 98°C → 20sec
  3. 66°C → 5sec
  4. 72°C → 30sec
  5. 72°C → 1min
  6. 4°C → hold


7/13

  • Ran 5ul of PCR with ladder, cancer construct and vaccine construct → to diagnose problem with gel
  • Miniprep of Dean vector (3ml LB culture)
  • Purification of PCR product → nanodropped 16.4ug/ul
  • Nanodrop of:
  1. Yellow 7/13 construct NEB purified → 16.4ng/ml
  2. Blue gel purification → 48.7ng/ul
  3. Pink PCR purification 7/13 → 102.2ng/ul
  4. 7/13 -RK Vector 1 → 48.2ng/ul
  5. 7/13 -RK Vector 2 → 96.3ng/ul


7/14

Project Progress

Ran a gel PCR of TDGF-1

Lanes:

  1. Ladder
  2. PCR1
  3. PCR2
  4. PCR3
  5. Digest
  6. Digest Control
PCR of TDGF-1 IDT Construct
  • 3 reactions set up with the same setup as test 5 from 6/30
  • Annealing Temperature at 66°C for 5 seconds
"Chocolate doesn't have a smell" -Ryan

BioBrick Ideas

  1. Smelly yeast → optimized from e. coli
  2. Twist on smelly e. coli on e. coli → regulated with quorum sensing
  3. OmpA/EnvZ recognize osmolarity
  4. Genetic Circuit
  5. Shine yellow light → Banana smell. Shine green light → wintergreen
  6. TetR operator system genetic circuit
  7. Have cell die at certain wavelength → killswitch


7/15

  • Ran a gel on PCR'ed Construct
  • 1st gel failed → Samara poured a gel onto Brian's gel
  • All 3 PCR products worked → purified PCR → 30ul PCR product in each tube → 150ul building buffer
Nanodrop
    PCR Tube Concentration (ug/ul)
    PCR #1 15.6
    PCR #2 12.3
    PCR #3 11.6
  • PCR IDT construct and 1 PCRed construct with 130.9 ug/ul concentration


7/17

  • Epoch purification of one of PCR products from 7/14
  • Same protocol at 7/15
  • Approx. 10ul of DNA in tube for PCR use
  • 30ul of elution buffer used
  • Elution step centrifuged for 2 minutes at 13000RPM
Ran a Nanodrop
    Content Concentration (ug/ul) 260/280
    Sample I 22.2 1.79
    Sample II 22.9 1.79
IDT Construct Digest (10ul Reaction)
    Content Experiment (ul) Control (ul)
    H2O 3.5 4.5
    10X Cutsmart 1 1
    XhoI .5 X
    PvoII-HF .5 X
    DNA (22.2ug/ul) 4.5 4.5
YEP352GAP Vector Digest (10ul Reaction)
    Content Experiment (ul) Control (ul)
    H2O 6.9 7.9
    10X Cutsmart 1 1
    XhoI .5 X
    PvoII-HF .5 X
    DNA (22.2ug/ul) 1.04 1.04
  • Digested at 37°C in incubator for an hour
  • 2ul of 6X Purple Loading Dye added to each of the four tubes after an hour digest to inactivate enzyme
Ran a gel

Lane:

  1. Brian's Construct
  2. Construct Digest - Control
  3. Construct Digest - Experimental
  4. Ladder
  5. Vector Digest - Experiment
  6. Vector Digest -Control
  • Ran at 116V for 30 minutes