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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 for AMO with USER endings 1.3.2 PCR for extraction of Nir1 1.3.3 Comment 1.4 Results 1.4.1 Gel on yesterdays PCR 1.4.2 Gel on ON PCR samples 1.4.3 Gel on todays PCR samples 1.5 Conclusion 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 2.5 Conclusion

Lab 208

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Main purpose

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• PCR for AMO with USER endings.
• Extraction PCR of Nir1

Who was in the lab

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Kristian, Julia, Henrike

Procedure

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PCR for AMO with USER endings

primers: 17a, 17b

template: gel purified AMO extraction fragment

Program: Standard with 54C annealing temperature and 3:00 extension time

Reaction Mix

component(per reaction)	without additives	using 5% DMSO	using 1M Betaine
dNTPs	1uL	1uL	1uL
HF buffer	10uL	10uL	10uL
X7 polymerase	0.5uL	0.5uL	0.5uL
MilliQ water	31.5uL	29uL	21.5uL
template	1uL	1uL	1uL
FW primer	3uL	3uL	3uL
RV primer	3uL	3uL	3uL
DMSO	-	2.5uL	-
Betaine	-	-	10uL

PCR for extraction of Nir1

primers: 41a, 41b

template: colony from plate. One colony was dissolved in 100 uL of MilliQ and 1 uL of this solution was taken as template

program: Touchdown PCR analog to program used on 01-08-2013 to amplify Nir.

Reaction Mix

component(per reaction)	without additives	using 5% DMSO	using 1M Betaine
dNTPs	1uL	1uL	1uL
HF buffer	10uL	10uL	10uL
X7 polymerase	0.5uL	0.5uL	0.5uL
MilliQ water	31.5uL	29uL	21.5uL
template	1uL	1uL	1uL
FW primer	3uL	3uL	3uL
RV primer	3uL	3uL	3uL
DMSO	-	2.5uL	-
Betaine	-	-	10uL

Comment

Both PCRs mentioned above were successful when DMSO was added.

Results

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Gel on yesterdays PCR

• 1kb ladder
• Yesterdays sample 1
• Yesterdays sample 2
• Yesterdays sample 3
• Yesterdays sample 4
• Neg.
• Neg.
• Nir2
• cycAX Histag
• 1kb ladder

Gel on ON PCR samples

• 1kb ladder
• Nir1
• Nir1 5%DMSO
• Nir1 1M Betaine
• Nir1 3M Betaine
• Nir2 3M Betaine U
• Nir2 1M Betaine U
• Nir2 5%DMSO U
• Nir2 U
• Neg (Nir)
• Ref 0%DMSO
• Ref 2%DMSO
• Ref 5%DMSO
• AMO U
• AMO 2%DMSO U
• Neg (AMO)
• NirG 5%DMSO
• NirG
• 1kb ladder

Gel on todays PCR samples

• 1kb ladder
• Nir Q5 poly
• Nir 2%DMSO Q5 poly
• Nir 3M Betaine Q5 poly
• Nir 5%DMSO Q5 poly
• Nir1 Q5 poly
• Nir1 2%DMSO Q5 poly
• Nir1 5%DMSO Q5 poly
• Nir1 3M Betaine Q5 poly
• AMO USER primers
• AMO +DMSO USER primers
• AMO +Betaine USER primers
• Nir1 DMSO
• Nir1 Betaine
• 1kb ladder

Conclusion

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Today's PCRs on AMO and Nir were successful when DMSO was added.

Lab 115

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Main purpose

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Run Experiment 2. E.coli control measurement.

Who was in the lab

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Ariadni, Natalia

Procedure

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Adjusting the temperature at 37 degrees and calibrating the probes as described in Appendix 5.

Following the protocol Experiment 2

Changing the steps :

19. Add 0.5 ml of nitrite solution and continue by adding 0.5 ml after 10 minutes.

We added 0.5 ml nitrite again and then we took 2 ml of sample, we added then 0.5 ml and afterwards when there was any change in the curve we spiked with 0.4 ml of nitrite. We took 2 ml for sample in the end and we added 0.5 ml of N₂O and 1 ml of NO to see if the probes are working properly.

Results

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Colorimetric results

Measurements of standard solutions

Ammonium - 43.7 mg/L ( expected 39 mg/L)

Nitrite - < 2 mg/L (X10 diluted) -0.4 mg/L (with no dilution) (expected 0.5 mg/L)

Nitrate - 18.7 mg/L (X10 diluted) (expected 12 mg/L)

Measurement of samples

Ammonium

Measuring range 2-75 mg/L NH₄-N

start point- no signal

middle point- signal <2 mg/L

end point- signal 0.7 mg/L

Nitrate

Measuring range 1-25 mg/L NO₃-N

start point- no signal

middle point- signal <1 mg/L

end point- signal <1 mg/L

Nitrite

Measuring range 0.02-1 mg/L NO₂-N

start point- no signal

middle point- signal 0.43 mg/L

end point- signal 0.79 mg/L

Conclusion

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Problems with the NO probe and there were not proper results.

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