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Lab 208

Main purpose

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

Who was in the lab

Kristian, Julia, Henrike

Procedure

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

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

Comment

Both PCRs mentioned above were successful when DMSO was added.

Results

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

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

Lab 115

Main purpose

Run Experiment 2

Control measurement

Who was in the lab

Ariadni, Natalia

Procedure

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

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

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

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