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Team:DTU-Denmark/Notebook/28 August 2013
From 2013.igem.org
28 August 2013
Contents |
Lab 208
Main purpose
PCR to amplify pSB1C3 and pZA21::ara for Nir
Who was in the lab
Henrike, Kristian, Julia
Procedure
A gradient PCR was made for pSB1C3 and pZA21::ara for Nir.
The master mix contains:
- CG buffer,
- X7 polymerase,
- 2% of DMSO (1uL of 100%DMSO) per reaction
- 1mM MgCl2 (1uL of 50mM MgCl2) per reaction
| 1 | 2 | |
|---|---|---|
| Template | pSB1C3 | pZA21::ara |
| Primers | 57a + 57b | 58a + 58b |
| Temperature range | 58C-78C | 50C-65C |
| Elongation time per cycle | 1:30 min | 2 min |
Each reaction was run in one of the 12 different annealing temperatures within the range indicated in the table. We followed the standard PCR program.
Results
Conclusion
lab 115
Main purpose
Run Experiment 4 in two different samples aerobically in order to characterize the behavior of HAO transformants and the Nitrosomonas europaea strain.
Who was in the lab
Ariadni, Helen, Kasia
Procedure
Adjusting the temperature at 36 degrees and calibrating the probes as described in Calibration protocol.
Following the protocol Experiment 4
Growing:
- two replicates of 4 ml from HAO overnight culture in 50 ml of DM minimal medium with NH4Cl and 25 ul of hydroxylamine solution
- 4 ml from Nitrosomonas Europaea in 42 ml of ATCC media with 20 ul of hydroxylamine solution
- 4 ml from Nitrosomonas Europaea in 44 ml of ATCC media with 0.7 ml of ammonium solution
OD was measured OD=0.4017 and 0.3864 for the HAO mutant. 0.0822 and 0.0947. For Nitrosomonas Europaea was 0.0095 and 0.0084.
Results
Colorimetric results
Ranges
- Measuring range 5-150 mg/L NH4-N
- Measuring range 0.02-1 mg/L NO2-N
Standard solutions
- Ammonium - 66.6 mg/L (expected 39 mg/L)
- Nitrite - 0.72 mg/L (expected 0.5 mg/L)
| time (min) | nitrite HAO1(mg/L) | nitrite HAO2(mg/L) | Ammonium N.Europaea(mg/L) | nitrite N.Europaea |
|---|---|---|---|---|
| 0 | <0.02 | <0.02 | 0 | <0.02 |
| 6 | <0.02 | <0.02 | 0 | <0.02 |
| 13 | <0.02 | <0.02 | 0 | <0.02 |
| spike | <0.02 | <0.02 | 246 | <0.02 |
| 25 | <0.02 | <0.02 | 250 | <0.02 |
| 39 | <0.02 | <0.02 | 278 | <0.02 |
| 105 | <0.02 | <0.02 | 266 | <0.02 |
| 19 hours | <0.02 | <0.02 | 220 | <0.02 |
Note: there was some more nitrite for N.Europaea but it was still less than <0.02 mg/L at the end of the experiment
OD at the end of the experiment
OD( HAO1)=0.28 and OD(HAO2)=0.278. For the N.Europaea culture the OD was 0.1348 and 0.0114 for the solution with ammonium and for the one with hydroxylamine, respectively.
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