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Team:DTU-Denmark/Notebook/9 July 2013
From 2013.igem.org
Contents |
208
Main purpose
Run gel with 7 PCR samples of Nir operon from Pseudomonas aeruginosa and purified AMO, HAO and CYC from 08.07.2013.
Purification of Nir operon and Nanodrop measurement.
Set up new PCR reaction to extract Nir from Pseudomonas aeruginosa.
Who was in the lab
Ariadni, Henrike, Julia, Gosia
Procedure
Run gel
Purification of Nir DNA according to the protocol QIA purification protocol. (given in the Kit)
Extraction PCR
to produce the right fragment of the Nir operon from either chromosomal DNA from Pseudomonas aeruginosa or the too long PCR fragments from the 07.07.
6 samples
- 3 samples from culture pAO1
- 3 samples from purification of the PCR that was done on 08.07.
master mix for 7 reactions
- dNTP: 1 ul (7 ul)
- HF buffer: 10 ul (70 ul)
- Phusion polymerase: 0.5 ul (3.5 ul)
- H2O: 31.5 ul (220.5 ul)
Settings for PCR
| Temperature (oC) | Time (min) | Rounds |
|---|---|---|
| 98 | 2:00 | 1 |
| 98 | 0:20 | 35 |
| 66 | 0:45 | 35 |
| 72 | 9:00 | 35 |
| 72 | 5:00 | 1 |
| 4 | ∞ | - |
Results
Nanodrop measurement of Nir operon:
- 17.98 ng/ul
- 16.82 ng/ul
- 8.51 ng/ul
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