Team:DTU-Denmark/Notebook/8 August 2013

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==Who was in the lab==
==Who was in the lab==
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Ariadni, Helen
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Ariadni, Natalia
==Procedure==
==Procedure==

Revision as of 11:57, 11 August 2013

8 August 2013

Contents

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

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



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

2013-08-08 unkown gel.jpg


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

2013-08-08 Nir.jpg


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

2013-08-08 nir1 AMO user.jpg

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, Natalia

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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