Team:DTU-Denmark/Notebook/9 July 2013

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(→Extraction PCR)
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===Extraction PCR===
===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.
+
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
6 samples
*3 samples from culture pAO1
*3 samples from culture pAO1
-
*3 samples from purification of the PCR that was done on 08.07.
+
*3 samples from today's purification of the PCR that was done on 08.07.
master mix for 7 reactions  
master mix for 7 reactions  
* dNTP: 1 ul (7 ul)
* dNTP: 1 ul (7 ul)

Revision as of 14:05, 18 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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