Difference between revisions of "Team:Manchester/Notebook"
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<b>Table 1. Master mix</b> | <b>Table 1. Master mix</b> | ||
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<li>Six different glucose concentrations were then made as depicted in table 2</li> | <li>Six different glucose concentrations were then made as depicted in table 2</li> | ||
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<b>Table 2. Glucose concentration</b> | <b>Table 2. Glucose concentration</b> | ||
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Revision as of 22:47, 13 October 2016
Notebook
Inducible Gene Switch
- Re-suspended DNA (constitutive promoters) from iGEM Distribution kit and transformed them into DH5α strain.
- Growth was found on the negative control plates so re-transformed DNA
- Inoculated of transformed cells.
Pilot Experiment
- Made stocks of our reagents Glucose Stock for 0.005 g/mL, Glucose Oxidase Stock for 0.0025 g/mL, HRP Stock for 250 μL/mL, ABTS for 0.0025 g/mL
Inducible Gene Switch
- Performed miniprep for overnight cultures
- Prepared fresh batch of DH5α chemical competent cells
- Restriction enzyme digest of constitutive promoters and control(pUC19) for validation with EcoRI and PstI
Pilot Experiment
- Still awaiting the arrival of the BMG spectrophotometer
Inducible Gene Switch
- Prepared DH5α chemical competent cells
- Prepared Chloramphenicol antibiotic stock
- Restreaked alcA 1 (BBa_K678001) and spispink(BBa_K1033923 pink chromoprotein)
Pilot Experiment
- We began by preparing master mix containing three reagents glucose oxidase, HRP and ABTS (table 1)
- Six different glucose concentrations were then made as depicted in table 2
- Protocol
- 50 μl of glucose solution was added into each well. Samples were run in triplicates
- Tube containing master mix was placed into spectrophotometer.
- Spectrophotometer was then used to measure absorbance of our green coloured product-oxidised ABTS at 420 nm. Absorbance values were taken every 2 sec for 3 min once 150 ul of master mix was added into each well.
- Results
- The rate of reaction did not level off and poor colour intensity was observed.
- We repeated the same experiment we did earlier this week. However instead of measuring absorbance for 3 min absorbance was measured every 2 sec for a 5 min period.
- Results - Reaction rate did level off after measuring absorbance for 5 min. However, colour intensity was still very poor.
| Final reagent (μg/ml) | |
|---|---|
| Glucose oxidase | 60 |
| HRP | 60 |
| ABTS | 100 |
| Final glucose concentration (μg/ml) |
|---|
| 0.50 |
| 1.00 |
| .25 |
| 1.50 |
| 1.75 |
| 2.00 |
