Team:DTU-Denmark/Notebook/27 August 2013

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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, Kashia

Procedure


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


Following the protocol Experiment 4

Changing the steps :

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

and 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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