IGEM:University of Chicago/2009/Notebook/Paraoxon Biosensor/2009/08/03

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August 3, 2009

  • Received primers for changing GFP to RFP/mcherry!
  • Diluted primers to 100 μM in TE buffer (spin down, add TE)
  • Let rest for 1-2 hours at room temperature
Concentrations of DNA + dilution:

template-RFP: 7.42 ng/μL diluted to 74 pg/μL
template-mcherry: 326 ng/μL diluted to 65.0 pg/μL
  • PCR
For both mcherry and RFPTurbo
beads: 2μL
5' primer: 5μL
3' primer: 5μL
template: 2μL
ddH2O: 36μL
Total: 50μL
Program name - RFP
Incubation: 95°C   5 minutes
Separation: 95°C   30 seconds
Annealing:  58°C   30 seconds
Extension:  72°C   1.5 minutes
Repeat x30
  • Fragments should be 1kbp long
  • Control 1 - No primers
  • Control 2 - no DNA (use RFP turbo primers and template)
2 beads
5' primers: 5μL
3' primers: 5μL
temp 2μL
ddH2O 46: 38μL
  • Ran a gel at 150V - should see band at ~750bp for mcherry and RFP turbo
  • gel will be inserted here!
  • very thick band seen at ~750
  • will do restriction digest/ligation tomorrow

August, 3rd, 2009 P2

OD660 Time 12:20 2:20 4:20 6:20 7:50 10:10 11:20
YGR035 Cont 0.186 0.222 .283 .242 0.436 0.882 1.213
YGR035 0.5 mM 0.183* 0.158 .137 .202 0.185 0.190 0.382
YGR346 Cont 0.171 0.301 .412 .974 1.252 1.383 1.478
YGR346 0.5mM 0.204* 0.289 .187 .223 0.277 0.270 0.444
1 Cont 0.366 0.416 .492 .975 1.331 1.452 1.427
1 0.5 mM 0.166* 0.408 .388 .618 1.029 1.218 1.346
2 Cont 0.168 0.337 .283 .324 0.490 0.898 1.223
2 0.5mM 0.189* 0.270 .396 .462 0.746 1.102 1.245
3 Cont 0.237 0.256 .247 .448 0.481 0.887 1.183
3 0.05 mM 0.274* 0.396 .330 .467 0.590 1.102 1.314
3 0.1 mM 0.126* 0.225 .263 .276 0.482 1.008 1.274
3 0.25 mM 0.239* 0.251 .232 .373 0.647 (0.697?) 1.140 1.305
3 0.5 mM 0.232* 0.349 .160 .281 0.408 0.720 1.053
4 Cont 0.229 0.265 .319 .362 0.371 0.586 0.959
4 0.5 mM 0.293* 0.236 .226 .281 0.319 0.236 0.277

Added to 4.5 ml cultures

             0.5 mM = 22 uL
             0.1 mM = 4.1 uL
             0.05 mM = 2.2 uL
             0.25 mM = 11 uL
  • = time of induction

Visual Test for Glow 1.(0.5) Inconclusive: no green glows

2.(0.5) ""

3. (0.1) ""

3. (0.25) ""

3. (0.5) ""

4. (0.5) ""

035 (0.5) ""

346 (0.5) ""

Glowing tests did not confirm any glowing yeast cultures.