Team:DTU-Denmark/Notebook/27 August 2013
From 2013.igem.org
27 August 2013
Contents |
lab 208
Main purpose
Who was in the lab
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.
Conclusion
lab 115
Main purpose
Run Experiment 4 in two different samples anaerobically in order to characterize the behavior of AMO and HAO transformants.
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 |
Conclusion
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