Team:Stony Brook/Notebook/Week3

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Week 3: 7/11-7/15

Week 3 (7/11-7//15)

7/11

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

Thermocycler Program
Step Temperature (Degrees C) Duration (seconds)
1 98 30
2 98 20
3 66 5
4 72 30
5 72 60
6 4 Hold
After step 4, return to step 2. Repeat steps 2-4 thirty-five times before continuing to step 5 and on.
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
Vaccine Group
  • Stuff & Things

7/12

Cancer group
  • Made gel for electrophoresis of PCR construct → it didn't work
  • Thermocycler Program: Same as PCR from 7/11

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
Vaccine Group
  • Stuff & Things

7/13

Cancer group
  • 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
Vaccine Group
  • Things

7/14

Cancer group
  • Things
Vaccine Group
Contents of well Amount (ul)
Ladder 10
AD 5
AO 5
AM 5
AN 5
Gal-Sunil Added 1ul loading dye
Con-Sunil 5
  • Brian and Sunil's Gal promoters worked but both constructs failed
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

Cancer group
  • Ran a gel on PCR products →
Vaccine group
  • Jon and Sunil retrying construct
  • Jon used phire at 50°C
  • Sunil used Q5. Extension at 72°C for 30 seconds
Polymerase Primers (ul) Template (ul) H2O (ul)
Phire (25ul) 2.5 Reverse Construct 2.5 Forward Stitch 1.5 19.5
Q5 (25ul) 2.5 Reverse Construct 2.5 Forward Stitch 1.5 19.5