lab 208

Main purpose

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

lab 115

Main purpose

Run experiment 2: "Measuring the production of N₂O from Nitrite NO₂^- anaerobically"

Control measurement

Who was in the lab

Ariadni, Helen

Procedure

Following the protocol "Experiment 2: Measuring production of N₂O from Nitrite NO₂^- anaerobically"

Changing the steps :

6. 2 ml of the overnight culture in 100 ml of medium.

7. Grow the cells for about 4 hours where the OD was measured ...... in 500 nm wavelength setup instead of 600 nm.

8. We didn't cool down the centrifuge because the experiment had to be done in 37 degrees.

11. The volume was 140 ml instead of 100 ml.

12. The OD was measured 0.1195 instead of 0.3.

15. 10 minutes of N₂ saturation instead of 5 minutes.

19. Add 0.5 ml of nitrite solution and continue by adding 1 ml after 10 minutes then we took 1.8 ml of sample, we added then 0.5 ml and afterwards when there was any change in the curve we spiked with 0.9 ml of nitrite. We took 2.3 ml for sample in the end and another 2 ml for OD measurement where OD=0.1105.

The temperature in the end was 40.5 degrees C and not 37.

Results

Colorimetric results

Ammonium

Measuring range 2-75 mg/L NH₄-N

start point- signal <2 mg/L

middle point- signal <2 mg/L

end point- signal <2 mg/L but 1.8 mg/L

Nitrate

Measuring range 1-25 mg/L NO₃-N

start point- signal <1 mg/L

middle point- signal <1 mg/L

end point- signal 0.4 mg/L

Nitrite

Measuring range 0.02-1 mg/L NO₂-N

start point- signal 0.07 mg/L

middle point- signal 0.57 mg/L after X10 dilution

end point- signal 0.47 mg/L after X10 dilution

Conclusion

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