Team:DTU-Denmark/Notebook/9 July 2013

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9_July_2013

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 08.07.

6 samples

  • 3 samples from culture pAO1
  • 3 samples from today's 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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