Team:DTU-Denmark/Notebook/28 August 2013

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==Main purpose==
==Main purpose==
<hr/>
<hr/>
 +
PCR to amplify pSB1C3 and pZA21::ara for Nir
==Who was in the lab==
==Who was in the lab==
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==Who was in the lab==
==Who was in the lab==
<hr/>
<hr/>
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Ariadni, Helen, Kashia
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Ariadni, Helen, Kasia
==Procedure==
==Procedure==
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* 4 ml from ''Nitrosomonas Europaea'' in 44 ml of ATCC media with 0.7 ml of ammonium solution
* 4 ml from ''Nitrosomonas Europaea'' in 44 ml of ATCC media with 0.7 ml of ammonium solution
   
   
 +
OD was measured OD=0.4017 and 0.3864 for the HAO mutant.  0.0822 and 0.0947. For ''Nitrosomonas Europaea'' was 0.0095 and 0.0084.
OD was measured OD=0.4017 and 0.3864 for the HAO mutant.  0.0822 and 0.0947. For ''Nitrosomonas Europaea'' was 0.0095 and 0.0084.
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{| class="wikitable" style="text-align: right"
{| class="wikitable" style="text-align: right"
-
! 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)
+
! time (min)!!nitrite HAO1(mg/L)!! nitrite HAO2(mg/L)!! Ammonium ''N.Europaea''(mg/L) !! nitrite ''N.Europaea''
|-
|-
-
|  0 || <5 || <5 || <5 || <0.02 || 5 || <0.02
+
|  0 || <0.02 || <0.02 || 0 || <0.02
|-
|-
-
| 13 || <5 || <5 || 5 || <0.02 || - || -
+
| 6 || <0.02 || <0.02 || 0 || <0.02
|-
|-
-
|  19 || <5 || <5 || 5 || <0.02 || - || -
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|  13 || <0.02 || <0.02 || 0 || <0.02
|-
|-
-
| spike || 146|| 140 || <5 || <0.02 || - || -
+
| spike || <0.02 || <0.02 || 246 || <0.02
|-
|-
-
|  35 || 158 || 244|| 258 || <0.02 || - || -
+
|  25 || <0.02 || <0.02 || 250 || <0.02
|-
|-
-
|  41 || 169.5 || 171 || 178 || <0.02 || - || -
+
|  39 || <0.02 || <0.02 || 278 || <0.02
|-
|-
-
|  52 || 226 || 280 || 250 || <0.02 || - || -
+
|  105 || <0.02 || <0.02 || 266 || <0.02
|-
|-
-
|  64 || 232 || 254 || 124 || <0.02 || - || -
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|  19 hours|| <0.02 || <0.02 || 220 || <0.02
-
|-
+
-
|  90|| 238 || 272  || 246 || <0.02 || - || -
+
-
|-
+
-
|  10 hours|| 172|| 218 || 16 || <0.02 || 154 || <0.02
+
|-
|-
|}
|}
 +
 +
Note: there was some more nitrite for ''N.Europaea'' but it was still less than <0.02 mg/L at the end of the experiment
===OD at the end of the experiment===
===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.
+
OD( HAO1)=0.28 and OD(HAO2)=0.278. For the ''N.Europaea'' culture the OD was 0.1348 and 0.0114 for the solution with ammonium and for the one with hydroxylamine, respectively.
 +
 
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==Conclusion==
 
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<hr/>
 
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Latest revision as of 12:08, 4 October 2013

28 August 2013

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Contents

Lab 208

Main purpose

PCR to amplify pSB1C3 and pZA21::ara for Nir

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 aerobically in order to characterize the behavior of HAO transformants and the Nitrosomonas europaea strain.

Who was in the lab

Ariadni, Helen, Kasia

Procedure

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


Following the protocol Experiment 4

Growing:

  • two replicates of 4 ml from HAO overnight culture in 50 ml of DM minimal medium with NH4Cl and 25 ul of hydroxylamine solution
  • 4 ml from Nitrosomonas Europaea in 42 ml of ATCC media with 20 ul of hydroxylamine solution
  • 4 ml from Nitrosomonas Europaea in 44 ml of ATCC media with 0.7 ml of ammonium solution


OD was measured OD=0.4017 and 0.3864 for the HAO mutant. 0.0822 and 0.0947. For Nitrosomonas Europaea was 0.0095 and 0.0084.

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)nitrite HAO1(mg/L) nitrite HAO2(mg/L) Ammonium N.Europaea(mg/L) nitrite N.Europaea
0 <0.02 <0.02 0 <0.02
6 <0.02 <0.02 0 <0.02
13 <0.02 <0.02 0 <0.02
spike <0.02 <0.02 246 <0.02
25 <0.02 <0.02 250 <0.02
39 <0.02 <0.02 278 <0.02
105 <0.02 <0.02 266 <0.02
19 hours <0.02 <0.02 220 <0.02

Note: there was some more nitrite for N.Europaea but it was still less than <0.02 mg/L at the end of the experiment

OD at the end of the experiment

OD( HAO1)=0.28 and OD(HAO2)=0.278. For the N.Europaea culture the OD was 0.1348 and 0.0114 for the solution with ammonium and for the one with hydroxylamine, respectively.


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