Team:DTU-Denmark/Notebook/28 August 2013

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28 August 2013

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Contents

Lab 208


Main purpose


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 anaerobically in order to characterize the behavior of AMO transformants.

Who was in the lab


Ariadni, Helen, Kashia

Procedure


Adjusting the temperature at 36 degrees and calibrating the probes as described in Calibration protocol.


Following the protocol Experiment 4

Changing the steps :

step 2 . 4 ml of the overnight culture growing in DM minimal medium with NH4Cl

and step 3 from the experimental procedure where the OD was measured OD=0.0761 and 0.0720 for the AMO mutant. For the E.coli culture the OD was 0.0822 and 0.0947.The abiotic controls have OD= 0.002 and 0.0006.

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)ammonium AMO1(mg/L) ammonium AMO2(mg/L) Ammonium E.coli(mg/L) nitrite E.coli ammonium abiotic (mg/L) nitrite abiotic(mg/L)
0 <5 <5 <5 <0.02 5 <0.02
13 <5 <5 5 <0.02 - -
19 <5 <5 5 <0.02 - -
spike 146 140 <5 <0.02 - -
35 158 244 258 <0.02 - -
41 169.5 171 178 <0.02 - -
52 226 280 250 <0.02 - -
64 232 254 124 <0.02 - -
90 238 272 246 <0.02 - -
10 hours 172 218 16 <0.02 154 <0.02

OD at the end of the experiment

OD=0.2083 and 0.2232 for the AMO mutants. For the E.coli culture the OD was 2.39 and 0.1173.The abiotic controls have OD=0.00144 and 0.0004.

Conclusion


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