Team:DTU-Denmark/Notebook/22 August 2013

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{{:Team:DTU-Denmark/Templates/StartPage|22 August 2013}}
{{:Team:DTU-Denmark/Templates/StartPage|22 August 2013}}
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Navigate to the [[Team:DTU-Denmark/Notebook/21_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/23_August_2013|Next]] Entry
=lab 208=
=lab 208=
<hr/>
<hr/>
==Main purpose==
==Main purpose==
<hr/>
<hr/>
 +
*USER reaction
 +
*PCR for biobrick parts
 +
*gel purification of pZA21::ara with USER endings
 +
*colony PCR
==Who was in the lab==
==Who was in the lab==
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Gel purified the linearized plasmid pZA21::ara with USER endings
Gel purified the linearized plasmid pZA21::ara with USER endings
-
===colony pPCR to verify AMO insert===
+
===colony PCR to verify AMO insert===
Picked 10 colonies from yesterday's cloning for colony PCR. Diluted cells in 50 uL of MilliQ and took 1 uL as template
Picked 10 colonies from yesterday's cloning for colony PCR. Diluted cells in 50 uL of MilliQ and took 1 uL as template
Line 89: Line 93:
<hr/>
<hr/>
 +
Purification of plasmids containing the araBAD SPL and RFP successful but with varying concentration.
=lab 115=
=lab 115=
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==Main purpose==
==Main purpose==
<hr/>
<hr/>
-
Run [[Team:DTU-Denmark/Experiment2|Experiment 2]] anaerobically in order to characterize the behavior of ''E.coli''.
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Run [[Team:DTU-Denmark/Experiment2|Experiment 2]] in two different samples anaerobically in order to characterize the behavior of ''E.coli''.
==Who was in the lab==
==Who was in the lab==
<hr/>
<hr/>
-
Ariadni, Helen, Natalia
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Ariadni, Helen
==Procedure==
==Procedure==
<hr/>
<hr/>
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Adjusting the temperature at 37 degrees and calibrating the probes as described in Appendix 5.
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Adjusting the temperature at 36 degrees and calibrating the probes as described in [[Team:DTU-Denmark/Methods/Calibrating_Electrodes|Calibration protocol]].
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Changing the steps :
Changing the steps :
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6. 3 ml of the overnight culture (from glycerol stock) in 200 ml of medium with OD=0.0317.
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6. 4 ml of the overnight culture growing in DM minimal medium
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7. Then added 3 ml more after 2 hours and the OD was 0.1046. The OD after 1 hour was 0.1340. Afterwards we added glycerol 1g in 10 ml of water and then added in our growing culture. Then after 40 min the OD was 0.2737. (Setup wavelength 600 nm).
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12. The OD was measured OD=0.261 in sample A and OD=0.263 in sample B.
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12. The OD was measured 0.1324 instead of 0.3.
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19.
 +
* 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.
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19. Add 0.5 ml of nitrite solution and continue by adding 0.5 ml after 10 minutes then we took 2.2 ml of sample, we added then 1 ml and we saw a slow response after 6 minutes. Afterwards when there was any change in the curve we spiked with 1 ml of nitrite. We continued as there was no response by adding 1 ml more and then by adding 2 ml more. We took 2 ml for sample in the end and another 2 ml for OD measurement where OD=0.1350. One hour after we took 2 ml of sample and then we spiked with 1 ml of NO<sub>2</sub>. There was no response after 12 minutes. Then we spiked with 0.5 ml of N<sub>2</sub>O and we show a big response.
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* 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.
==Results==
==Results==
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===Colorimetric results===
===Colorimetric results===
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'''Ammonium'''
 
-
''Measuring range 2-75 mg/L NH<sub>4</sub>-N''
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'''Ranges'''
 +
* ''Measuring range 2-75 mg/L NH<sub>4</sub>-N''
 +
* ''Measuring range 1-25 mg/L NO<sub>3</sub>-N''
 +
* ''Measuring range 0.02-1 mg/L NO<sub>2</sub>-N''
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start point- signal <2 mg/L
 
-
middle point- signal 2 mg/L
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Standard solutions
 +
* Ammonium - 66.6 mg/L (expected 39 mg/L)
 +
* Nitrite - 0.72 mg/L (expected 0.5 mg/L)
 +
* Nitrate -
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end point- signal <2 mg/L
 
 +
'''For Sample A'''
 +
{| class="wikitable" style="text-align: right"
 +
! time !! nitrite (mg/L)!! nitrate (mg/L) !! ammonium (mg/L)
 +
|-
 +
|  0 || <0.02 || <1 || 3.8
 +
|-
 +
|  5 || <0.02 || <1 || <2
 +
|-
 +
|  10|| <0.02 || <1 || 0.3
 +
|-
 +
| spike || 0.7 || <1 || <2
 +
|-
 +
|  15 || 0.7 || <1 || 0.7
 +
|-
 +
|  20 || 0.71 || <1 || 0.2
 +
|-
 +
|  25 || 0.72 || <1 || <2
 +
|-
 +
|  second spike || 1.46 || 1.7 ||2.7
 +
|-
 +
|  35|| 1.42 || 1.6 || <2
 +
|-
 +
|}
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'''Nitrate'''
+
'''For Sample B'''
 +
{| class="wikitable" style="text-align: right"
 +
! time !! nitrite (mg/L)!! nitrate (mg/L) !! ammonium (mg/L)
 +
|-
 +
|  0 || <0.02 || - || <2
 +
|-
 +
|  5 || <0.02 || - || <2
 +
|-
 +
|  10|| <0.02 || - || <2
 +
|-
 +
| spike || 0.36 || - || <2
 +
|-
 +
|  15 || 0.4 || - || <2
 +
|-
 +
|  20 || 0.4 || <1 || <2
 +
|-
 +
|  second spike || 2.12 || 2.3 || <2
 +
|-
 +
|  30 || 1.78 || 2.6 || 0.4
 +
|-
 +
|  35 || 1.74 || 2.7 || <2
 +
|-
 +
|}
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''Measuring range 1-25 mg/L NO<sub>3</sub>-N''
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=== N<sub>2</sub>O measurement===
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start point- signal <1 mg/L
 
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middle point- signal <1 mg/L but visible pinker
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[[File:Dtu Experiment 2 round 2 --sample A.png|600px]]
 +
[[File:Dtu Experiment 2 round 2 -- sample B.png|600px]]
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end point- signal <1 mg/L but much visible pinker
+
==Conclusion==
 +
There was no response from native ''E.coli''.
-
'''Nitrite'''
 
-
 
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''Measuring range 0.02-1 mg/L NO<sub>2</sub>-N''
 
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start point- signal <0.02 mg/L
 
-
 
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middle point- signal 0.29 mg/L
 
-
 
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end point- signal 0.70 mg/L after X2 dilution
 
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-
 
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Point before spiking with NO<sub>2</sub> at the end
 
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measurement 1.03 mg/L (x2 dilution)- 0.5 mg/L (x4 dilution)
 
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==Conclusion==
 
-
<hr/>
 
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The ''E.coli'' didn't grow as usual and the problem might be the minimal medium.
 

Latest revision as of 17:53, 28 September 2013

22 August 2013

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Contents

lab 208

Main purpose

  • USER reaction
  • PCR for biobrick parts
  • gel purification of pZA21::ara with USER endings
  • colony PCR

Who was in the lab

Kristian, Henrike

Procedure

USER ligation and transformation

Redid for HAO and cyc in arabinose inducible pZA21::araBAD and for Nir fragments

PCR for Biobrick parts

Set up a new PCR reaction for Biobrick parts using HF buffer and 5% DMSO but no MgCl2. PCR was run on a touchdown program

primers: 53a, 53b

template: Sec2 miniprep

program:

temperature time cycles
98C 2:00 -
98C 0:10 10
63C 1:00 10
-0.5C per cycle
72C 1:00 10
98C 0:10 25
53C 1:00 25
72C 1:00 25
72C 5:00 -
10C hold -

Gel purification

Gel purified the linearized plasmid pZA21::ara with USER endings

colony PCR to verify AMO insert

Picked 10 colonies from yesterday's cloning for colony PCR. Diluted cells in 50 uL of MilliQ and took 1 uL as template

primers: 53a, 53b

program:

temperature time cycles
98C 10:00 -
98C 0:10 36
56C 0:30 36
72C 2:00 36
72C 5:00 -
10C hold -

Results

nanodrop measurement of ara spl plasmids

Nd ara spl.jpg

Conclusion

Purification of plasmids containing the araBAD SPL and RFP successful but with varying concentration.

lab 115

Main purpose

Run Experiment 2 in two different samples anaerobically in order to characterize the behavior of E.coli.

Who was in the lab

Ariadni, Helen

Procedure

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


Following the protocol Experiment 2

Changing the steps :

6. 4 ml of the overnight culture growing in DM minimal medium

12. The OD was measured OD=0.261 in sample A and OD=0.263 in sample B.

19.

  • 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.

Results

Colorimetric results

Ranges

  • Measuring range 2-75 mg/L NH4-N
  • Measuring range 1-25 mg/L NO3-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)
  • Nitrate -


For Sample A

time nitrite (mg/L) nitrate (mg/L) ammonium (mg/L)
0 <0.02 <1 3.8
5 <0.02 <1 <2
10 <0.02 <1 0.3
spike 0.7 <1 <2
15 0.7 <1 0.7
20 0.71 <1 0.2
25 0.72 <1 <2
second spike 1.46 1.7 2.7
35 1.42 1.6 <2

For Sample B

time nitrite (mg/L) nitrate (mg/L) ammonium (mg/L)
0 <0.02 - <2
5 <0.02 - <2
10 <0.02 - <2
spike 0.36 - <2
15 0.4 - <2
20 0.4 <1 <2
second spike 2.12 2.3 <2
30 1.78 2.6 0.4
35 1.74 2.7 <2

N2O measurement

Dtu Experiment 2 round 2 --sample A.png Dtu Experiment 2 round 2 -- sample B.png

Conclusion

There was no response from native E.coli.



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