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Contents 1 lab 208 1.1 Main purpose 1.2 Who was in the lab 1.3 Procedure 1.3.1 PCR reactions 1.4 Results 1.4.1 Gel 2 lab 115 2.1 Main purpose 2.2 Who was in the lab 2.3 Procedure 2.4 Results 2.4.1 Colorimetric results 2.4.2 OD at the end of the experiment

lab 208

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Main purpose

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various PCR reactions

Who was in the lab

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Kristian, Henrike

Procedure

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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

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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

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Main purpose

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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

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Ariadni, Helen, Kasia

Procedure

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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

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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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