"
Team:DTU-Denmark/Notebook/27 August 2013
From 2013.igem.org
(→Colorimetric results) |
(→Procedure) |
||
| (9 intermediate revisions not shown) | |||
| Line 5: | Line 5: | ||
==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== | ||
| Line 23: | Line 25: | ||
pSB1C3: | pSB1C3: | ||
{| class="wikitable" style="text-align: right" | {| class="wikitable" style="text-align: right" | ||
| - | ! temperature !! time !! cycles | + | ! 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]] | ||
| - | |||
| - | |||
| - | |||
| - | |||
=lab 115= | =lab 115= | ||
| Line 196: | Line 194: | ||
==Main purpose== | ==Main purpose== | ||
<hr/> | <hr/> | ||
| - | Run [[Team:DTU-Denmark/Experiment2|Experiment 4]] in two different samples | + | 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, | + | Ariadni, Helen, Kasia |
==Procedure== | ==Procedure== | ||
<hr/> | <hr/> | ||
| - | Adjusting the temperature | + | 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 4]], with the changes in the following steps: | ||
| - | + | * 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. | |
| - | + | ||
| - | + | ||
| - | step 2 | + | |
| - | + | ||
| - | + | ||
==Results== | ==Results== | ||
| Line 223: | Line 217: | ||
'''Ranges''' | '''Ranges''' | ||
* ''Measuring range 5-150 mg/L NH<sub>4</sub>-N'' | * ''Measuring range 5-150 mg/L NH<sub>4</sub>-N'' | ||
| - | |||
* ''Measuring range 0.02-1 mg/L NO<sub>2</sub>-N'' | * ''Measuring range 0.02-1 mg/L NO<sub>2</sub>-N'' | ||
| Line 230: | Line 223: | ||
* 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) | ||
| - | |||
| Line 261: | Line 253: | ||
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=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
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.
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.
Navigate to the Previous or the Next Entry
