Team:DTU-Denmark/Notebook/8 August 2013

From 2013.igem.org

(Difference between revisions)
(→Gel on ON PCR samples)
(→Procedure)
 
(33 intermediate revisions not shown)
Line 1: Line 1:
{{:Team:DTU-Denmark/Templates/StartPage|8 August 2013}}
{{: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/>
<hr/>
==Main purpose==
==Main purpose==
<hr/>
<hr/>
 +
*PCR for AMO with USER endings.
 +
*Extraction PCR of Nir1
==Who was in the lab==
==Who was in the lab==
Line 13: Line 15:
<hr/>
<hr/>
-
==Results==
+
===PCR for AMO with USER endings===
-
<hr/>
+
 +
primers: 17a, 17b
 +
template: gel purified AMO extraction fragment
 +
 +
Program: Standard with 54C annealing temperature and 3:00 extension time
 +
 +
Reaction Mix
 +
{| class="wikitable" style="text-align: right"
 +
! 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 [[Team:DTU-Denmark/Notebook/1_August_2013| 01-08-2013]] to amplify Nir.
 +
 +
Reaction Mix
 +
{| class="wikitable" style="text-align: right"
 +
! 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==
 +
<hr/>
===Gel on yesterdays PCR===
===Gel on yesterdays PCR===
Line 54: Line 122:
*1kb ladder
*1kb ladder
[[File:2013-08-08 Nir.jpg| 600px]]
[[File:2013-08-08 Nir.jpg| 600px]]
 +
 +
 +
===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
 +
[[File:2013-08-08 nir1 AMO user.jpg| 600px]]
 +
 +
==Conclusion==
 +
<hr/>
 +
Today's PCRs on AMO and Nir were successful when DMSO was added.
 +
 +
=Lab 115=
 +
<hr/>
 +
 +
==Main purpose==
 +
<hr/>
 +
Run [[Team:DTU-Denmark/Experiment2|Experiment 2]]. ''E.coli'' control measurement.
 +
 +
==Who was in the lab==
 +
<hr/>
 +
Ariadni, Natalia
 +
 +
==Procedure==
 +
<hr/>
 +
 +
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 :
 +
 +
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 N<sub>2</sub>O and 1 ml of NO to see if the probes are working properly.
 +
 +
==Results==
 +
<hr/>
 +
 +
===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 NH<sub>4</sub>-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 NO<sub>3</sub>-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 NO<sub>2</sub>-N''
 +
 +
start point- no signal
 +
 +
middle point- signal 0.43 mg/L
 +
 +
end point- signal 0.79 mg/L
==Conclusion==
==Conclusion==
<hr/>
<hr/>
-
1% agarose gel
+
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
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/Templates/EndPage}}

Latest revision as of 14:45, 1 October 2013

8 August 2013

Navigate to the Previous or the Next Entry

Contents

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

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

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

Retrieved from "http://2013.igem.org/Team:DTU-Denmark/Notebook/8_August_2013"