Team:DTU-Denmark/Notebook/27 August 2013

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==Main purpose==
==Main purpose==
<hr/>
<hr/>
 +
various PCR reactions
==Who was in the lab==
==Who was in the lab==
<hr/>
<hr/>
 +
Kristian, Henrike
==Procedure==
==Procedure==
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pSB1C3:
pSB1C3:
{| class="wikitable" style="text-align: right"
{| class="wikitable" style="text-align: right"
-
! temperature !! time !! cycles
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! temperature !! time !! cycles  
|-
|-
|  98C || 2:00 || -  
|  98C || 2:00 || -  
Line 186: Line 188:
[[File:2013-08-27 bb pzaarafornir.jpg|600px]]
[[File:2013-08-27 bb pzaarafornir.jpg|600px]]
-
 
-
==Conclusion==
 
-
<hr/>
 
-
 
=lab 115=
=lab 115=
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==Main purpose==
==Main purpose==
<hr/>
<hr/>
-
Run [[Team:DTU-Denmark/Experiment2|Experiment 4]] in two different samples anaerobically in order to characterize the behavior of ''AMO and HAO transformants''.
+
Run [[Team:DTU-Denmark/Experiment2|Experiment 4]] in two different samples aerobically in order to characterize the behavior of ''AMO transformants'' and the '''''E.coli''''' strain.
==Who was in the lab==
==Who was in the lab==
<hr/>
<hr/>
-
Ariadni, Helen, Kashia
+
Ariadni, Helen, Kasia
==Procedure==
==Procedure==
<hr/>
<hr/>
-
Adjusting the temperature at 36 degrees and calibrating the probes as described in [[Team:DTU-Denmark/Methods/Calibrating_Electrodes|Calibration protocol]].
+
Adjusting the temperature to 37 degrees and calibrating the probes is described in [[Team:DTU-Denmark/Methods/Calibrating_Electrodes|Calibration protocol]].
-
 
+
-
 
+
-
Following the protocol [[Team:DTU-Denmark/Methods/Determining_concentration_of_nitrogen_compounds/Experiment_4|Experiment 2]]
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-
 
+
-
Changing the steps :
+
-
 
+
-
6. 4 ml of the overnight culture growing in DM minimal medium
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-
 
+
-
12. The OD was measured OD=0.261 in sample A and OD=0.263 in sample B.
+
-
19.
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Following the protocol [[Team:DTU-Denmark/Methods/Determining_concentration_of_nitrogen_compounds/Experiment_4|Experiment 4]], with the changes in the following steps:
-
* For sample A after taking the first sample, we continue with two more samples after 5 and 10 minutes. Then we add 2 ml of nitrite solution and continue by taking a sample after spiking. Then we took three more samples and after 15 minutes we spike with 2 ml of nitrite, and finally we took a last sample. The final OD measurement was OD=0.244 and the temperature was 36.8 degrees.
+
-
* For sample B after taking the first sample, we continue with two more samples after 5 and 10 minutes. Then we add 1 ml of nitrite solution and continue by taking a sample after spiking. Then we took two more samples and after 10 minutes we spike with 4 ml of nitrite, and then there was a response after 12 minutes were finally we took one sample and after 5 minutes the last. The final OD measurement was OD=0.248 and the temperature was 36.4 degrees.
+
* step 2: 4 ml of the overnight culture growing in DM minimal medium with NH<sub>4</sub>Cl
 +
* 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==
==Results==
Line 227: Line 216:
'''Ranges'''
'''Ranges'''
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* ''Measuring range 2-75 mg/L NH<sub>4</sub>-N''
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* ''Measuring range 5-150 mg/L NH<sub>4</sub>-N''
-
* ''Measuring range 1-25 mg/L NO<sub>3</sub>-N''
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* ''Measuring range 0.02-1 mg/L NO<sub>2</sub>-N''
* ''Measuring range 0.02-1 mg/L NO<sub>2</sub>-N''
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* Ammonium - 66.6 mg/L (expected 39 mg/L)
* Ammonium - 66.6 mg/L (expected 39 mg/L)
* Nitrite - 0.72 mg/L (expected 0.5 mg/L)
* Nitrite - 0.72 mg/L (expected 0.5 mg/L)
-
* Nitrate -
 
-
'''For Sample A'''
 
{| class="wikitable" style="text-align: right"
{| class="wikitable" style="text-align: right"
-
! time !! nitrite (mg/L)!! nitrate (mg/L) !! ammonium (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 || <0.02 || <1 || 3.8
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|  0 || <5 || <5 || <5 || <0.02 || 5 || <0.02
|-
|-
-
|  5 || <0.02 || <1 || <2
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|  13 || <5 || <5 || 5 || <0.02 || - || -
|-
|-
-
|  10|| <0.02 || <1 || 0.3
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|  19 || <5 || <5 || 5 || <0.02 || - || -
|-
|-
-
| spike || 0.7 || <1 || <2
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| spike || 146|| 140 || <5 || <0.02 || - || -
|-
|-
-
|  15 || 0.7 || <1 || 0.7
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|  35 || 158 || 244|| 258 || <0.02 || - || -
|-
|-
-
|  20 || 0.71 || <1 || 0.2
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|  41 || 169.5 || 171 || 178 || <0.02 || - || -
|-
|-
-
|  25 || 0.72 || <1 || <2
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|  52 || 226 || 280 || 250 || <0.02 || - || -
|-
|-
-
|  second spike || 1.46 || 1.7 ||2.7 ||
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|  64 || 232 || 254 || 124 || <0.02 || - || -
|-
|-
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|  35|| 1.42 || 1.6 || <2
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|  90|| 238 || 272  || 246 || <0.02 || - || -
|-
|-
-
|}
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|  10 hours|| 172|| 218 || 16 || <0.02 || 154 || <0.02
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'''For Sample B'''
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{| class="wikitable" style="text-align: right"
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! time !! nitrite (mg/L)!! nitrate (mg/L) !! ammonium (mg/L)
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-
|-
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-
|  0 || <0.02 || - || <2
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-
|-
+
-
|  5 || <0.02 || - || <2
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-
|-
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-
|  10|| <0.02 || - || <2
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-
|-
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| spike || 0.36 || - || <2
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-
|-
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|  15 || 0.4 || - || <2
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-
|-
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|  20 || 0.4 || <1 || <2
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-
|-
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|  second spike || 2.12 || 2.3 || <2
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-
|-
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|  30 || 1.78 || 2.6 || 0.4
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-
|-
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|  35 || 1.74 || 2.7 || <2
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|-
|-
|}
|}
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==Conclusion==
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===OD at the end of the experiment===
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<hr/>
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 +
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.

Latest revision as of 14:33, 4 October 2013

27 August 2013

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Contents

lab 208

Main purpose

various PCR reactions

Who was in the lab

Kristian, Henrike

Procedure

PCR reactions

Set up PCR reactions for:

  • Biobrick backbone pSB1C3 with USER endings fitting for our inserts
  • constitutive reference promoter in pZA21 (for SPL)
  • pZA21::ara vector with USER endings fitting for Nir1 and Nir2 inserts

first round of PCRs:

pSB1C3:

temperature time cycles
98C 2:00 -
98C 0:10 36
70C 0:45 36
72C 1:30 36
72C 5:00 -
10C hold -

Four different reaction mixes: HF, HF+5%DMSO, GC, GC+5%DMSO

pZA21::ara for Nir:

temperature time cycles
98C 2:00 -
98C 0:10 36
60C 1:00 36
72C 2:00 36
72C 5:00 -
10C hold -

Standard reaction mix.

constitutive reference promoter:

temperature time cycles
98C 2:00 -
98C 0:20 36
58.1C 1:00 36
72C 3:00 36
72C 5:00 -
10C hold -

Mix with GC, 5%DMSO and 2uL MgCl2.

pSB1C3 and pZA21::ara for Nir were without results (see gel picture). New PCRs were set up with one standard reaction and one reaction with additives. The annealing temperature was lowered.

second round of PCRs:

compound amount (in uL)
standard mix additive mix
dNTPs 1 1
X7 ploymerase 0.5 0.5
HF buffer 10 10
MilliQ 31.5 27
FW primer 3 3
RV primer 3 3
template 1 1
DMSO - 2.5
50 mM MgCl2 - 2


pSB1C3:

temperature time cycles
98C 2:00 -
98C 0:10 36
60C 1:00 36
72C 1:30 36
72C 5:00 -
10C hold -


pZA21::ara for Nir:

temperature time cycles
98C 2:00 -
98C 0:10 36
55C 1:00 36
72C 2:00 36
72C 5:00 -
10C hold -


Additionally set up a PCR for cycAX with USER endings since the amount of fragment is running low.

cycAX for USER:

temperature time cycles
98C 2:00 -
98C 0:20 36
57C 0:45 36
72C 1:30 36
72C 5:00 -
10C hold -

Standard reaction mix.

Results

Gel

  • 1 kb ladder
  • pSB1C3 for Biobricks, HF buffer
  • pSB1C3 for Biobricks, HF buffer, 5%DMSO
  • pSB1C3 for Biobricks, GC buffer
  • pSB1C3 for Biobricks, GC buffer, 5%DMSO
  • negative pSB1C3 for Biobricks
  • pZA21::ara for USER ligation with Nir
  • pZA21::ara for USER ligation with Nir (duplicate)
  • 1 kb ladder

Note: It seems the ladder overflew.

2013-08-27 bb pzaarafornir.jpg

lab 115

Main purpose

Run Experiment 4 in two different samples aerobically in order to characterize the behavior of AMO transformants and the E.coli strain.

Who was in the lab

Ariadni, Helen, Kasia

Procedure

Adjusting the temperature to 37 degrees and calibrating the probes is described in Calibration protocol.

Following the protocol Experiment 4, with the changes in the following steps:

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


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