"
Team:DTU-Denmark/Notebook/22 August 2013
From 2013.igem.org
22 August 2013
Contents |
lab 208
Main purpose
Who was in the lab
Kristian, Henrike
Procedure
USER ligation and transformation
Redid for HAO and cyc in arabinose inducible pZA21::araBAD and for Nir fragments
PCR for Biobrick parts
Set up a new PCR reaction for Biobrick parts using HF buffer and 5% DMSO but no MgCl2. PCR was run on a touchdown program
primers: 53a, 53b
template: Sec2 miniprep
program:
| temperature | time | cycles |
|---|---|---|
| 98C | 2:00 | - |
| 98C | 0:10 | 10 |
| 63C | 1:00 | 10 |
| -0.5C per cycle | ||
| 72C | 1:00 | 10 |
| 98C | 0:10 | 25 |
| 53C | 1:00 | 25 |
| 72C | 1:00 | 25 |
| 72C | 5:00 | - |
| 10C | hold | - |
Gel purification
Gel purified the linearized plasmid pZA21::ara with USER endings
colony pPCR to verify AMO insert
Picked 10 colonies from yesterday's cloning for colony PCR. Diluted cells in 50 uL of MilliQ and took 1 uL as template
primers: 53a, 53b
program:
| temperature | time | cycles |
|---|---|---|
| 98C | 10:00 | - |
| 98C | 0:10 | 36 |
| 56C | 0:30 | 36 |
| 72C | 2:00 | 36 |
| 72C | 5:00 | - |
| 10C | hold | - |
Results
nanodrop measurement of ara spl plasmids
Conclusion
lab 115
Main purpose
Run Experiment 2 in two different samples anaerobically in order to characterize the behavior of E.coli.
Who was in the lab
Ariadni, Helen
Procedure
Adjusting the temperature at 36 degrees and calibrating the probes as described in Calibration protocol.
Following the protocol Experiment 2
Changing the steps :
6. 4 ml of the overnight culture growing in DM minimal medium
12. The OD was measured OD=0.261 in sample A and OD=0.263 in sample B.
19.
- For sample A after taking the first sample, we continue with two more samples after 5 and 10 minutes. Then we add 2 ml of nitrite solution and continue by taking a sample after spiking. Then we took three more samples and after 15 minutes we spike with 2 ml of nitrite, and finally we took a last sample. The final OD measurement was OD=0.244 and the temperature was 36.8 degrees.
- For sample B after taking the first sample, we continue with two more samples after 5 and 10 minutes. Then we add 1 ml of nitrite solution and continue by taking a sample after spiking. Then we took two more samples and after 10 minutes we spike with 4 ml of nitrite, and then there was a response after 12 minutes were finally we took one sample and after 5 minutes the last. The final OD measurement was OD=0.248 and the temperature was 36.4 degrees.
Results
Colorimetric results
For Sample A
| time | nitrite (mg/L) | nitrate (mg/L) | ammonium (mg/L) | |
|---|---|---|---|---|
| 0 | <0.02 | <1 | 3.8 | |
| 5 | <0.02 | <1 | <2 | |
| 10 | <0.02 | <1 | 0.3 | |
| spike | 0.7 | <1 | <2 | |
| 15 | 0.7 | <1 | 0.7 | |
| 20 | 0.71 | <1 | 0.2 | |
| 25 | 0.72 | <1 | <2 | |
| second spike | 1.46 | 1.7 | 2.7 | |
| 35 | 1.42 | 1.6 | <2 |
For Sample B
| time | nitrite (mg/L) | nitrate (mg/L) | ammonium (mg/L) |
|---|---|---|---|
| 0 | <0.02 | - | <2 |
| 5 | <0.02 | - | <2 |
| 10 | <0.02 | - | <2 |
| spike | 0.36 | - | <2 |
| 15 | 0.4 | - | <2 |
| 20 | 0.4 | <1 | <2 |
| second spike | 2.12 | 2.3 | <2 |
| 30 | 1.78 | 2.6 | 0.4 |
| 35 | 1.74 | 2.7 | <2 |
Conclusion
The E.coli didn't grow as usual and the problem might be the minimal medium.
Navigate to the Previous or the Next Entry
