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Latest revision as of 14:45, 1 October 2013

8 August 2013

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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

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

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. E.coli control measurement.

Who was in the lab

Ariadni, Natalia

Procedure

Adjusting the temperature at 37<up>o</sup> 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.

Navigate to the Previous or the Next Entry

@@ Line 1: / Line 1: @@
 {{:Team:DTU-Denmark/Templates/StartPage|8 August 2013}}
+Navigate to the [[Team:DTU-Denmark/Notebook/7_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/9_August_2013|Next]] Entry
-=lab 208=
+=Lab 208=
 <hr/>
 ==Main purpose==
 <hr/>
+*PCR for AMO with USER endings.
+*Extraction PCR of Nir1
 ==Who was in the lab==
@@ Line 83: / Line 85: @@
 ==Results==
 <hr/>
 ===Gel on yesterdays PCR===
@@ Line 145: / Line 145: @@
 ==Conclusion==
 <hr/>
+Today's PCRs on AMO and Nir were successful when DMSO was added.
-=lab 115=
+=Lab 115=
 <hr/>
 ==Main purpose==
 <hr/>
-Run experiment 2: "Measuring the production of N<sub>2</sub>O from Nitrite NO<sub>2</sub><sup>-<sup>
+Run [[Team:DTU-Denmark/Experiment2|Experiment 2]]. ''E.coli'' control measurement.
-anaerobically in order to characterize the behavior of P.aeruginosa.
 ==Who was in the lab==
 <hr/>
-Ariadni, Helen
+Ariadni, Natalia
 ==Procedure==
 <hr/>
-Following the protocol "Experiment 2: Measuring production of N<sub>2</sub>O from Nitrite NO<sub>2</sub><sup>-</sup> anaerobically"
+Adjusting the temperature at 37<up>o</sup> and calibrating the probes as described in Appendix 5.
+Following the protocol [[Team:DTU-Denmark/Methods/Determining_concentration_of_nitrogen_compounds/Experiment_2|Experiment 2]]
 Changing the steps :
-. 2 ml of the overnight culture in 100 ml of medium.
+. Add 0.5 ml of nitrite solution and continue by adding 0.5 ml after 10 minutes.
-. Grow the cells for about 4 hours where the OD was measured ...... in 500 nm wavelength setup instead of 600 nm.
+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 N<sub>2</sub>O and 1 ml of NO to see if the probes are working properly.
-. We didn't cool down the centrifuge because the experiment had to be done in 37 degrees.
+==Results==
+<hr/>
-. The volume was 140 ml instead of 100 ml.
+===Colorimetric results===
-. The OD was measured 0.1195 instead of 0.3.
+Measurements of standard solutions
-. 10 minutes of N<sub>2</sub> saturation instead of 5 minutes.
+Ammonium - 43.7 mg/L ( expected 39 mg/L)
-. 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.
+Nitrite - < 2 mg/L (X10 diluted) -0.4 mg/L (with no dilution) (expected 0.5 mg/L)
-The temperature in the end was 40.5 degrees C and not 37.
+Nitrate - 18.7 mg/L (X10 diluted) (expected 12 mg/L)
+Measurement of samples
-==Results==
-<hr/>
-===Colorimetric results===
 '''Ammonium'''
 ''Measuring range 2-75 mg/L NH<sub>4</sub>-N''
-start point- signal <2 mg/L
+start point- no signal
 middle point- signal <2 mg/L
-end point- signal <2 mg/L but 1.8 mg/L
+end point- signal 0.7 mg/L
@@ Line 201: / Line 203: @@
 ''Measuring range 1-25 mg/L NO<sub>3</sub>-N''
-start point- signal <1 mg/L
+start point- no signal
 middle point- signal <1 mg/L
-end point- signal 0.4 mg/L
+end point- signal <1 mg/L
@@ Line 212: / Line 214: @@
 ''Measuring range 0.02-1 mg/L NO<sub>2</sub>-N''
-start point- signal 0.07 mg/L
+start point- no signal
-middle point- signal 0.57 mg/L after X10 dilution
+middle point- signal 0.43 mg/L
-end point- signal 0.47 mg/L after X10 dilution
+end point- signal 0.79 mg/L
 ==Conclusion==
 <hr/>
+Problems with the NO probe and there were not proper results.
 Navigate to the [[Team:DTU-Denmark/Notebook/7_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/9_August_2013|Next]] Entry
 {{:Team:DTU-Denmark/Templates/EndPage}}

Team:DTU-Denmark/Notebook/8 August 2013

From 2013.igem.org

Latest revision as of 14:45, 1 October 2013

8 August 2013

Contents

Lab 208

Main purpose

Who was in the lab

Procedure

PCR for AMO with USER endings

PCR for extraction of Nir1

Comment

Results

Gel on yesterdays PCR

Gel on ON PCR samples

Gel on todays PCR samples

Conclusion

Lab 115

Main purpose

Who was in the lab

Procedure

Results

Colorimetric results

Conclusion

Sponsors