Team:DTU-Denmark/Notebook/6 September 2013

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{{:Team:DTU-Denmark/Templates/StartPage|6 September 2013}}
{{:Team:DTU-Denmark/Templates/StartPage|6 September 2013}}
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Navigate to the [[Team:DTU-Denmark/Notebook/5_September_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/7_September_2013|Next]] Entry
Navigate to the [[Team:DTU-Denmark/Notebook/5_September_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/7_September_2013|Next]] Entry
=Lab 208=
=Lab 208=
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==Main purpose==
==Main purpose==
<hr/>
<hr/>
-
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*Add USER endings to HAO gene extracted from ''Nitrosomonas europaea''
==Who was in the lab==
==Who was in the lab==
<hr/>
<hr/>
-
 
+
Henrike
==Procedure==
==Procedure==
<hr/>
<hr/>
-
Template: HAO extraction fragment -isolated from 'Nitrosomonas europea'- (purified from PCR run on 04.09)
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 +
===Repeat HAO USER PCR from yesterday with same conditions and programs===
 +
 
 +
Template: HAO extraction fragment -isolated from ''Nitrosomonas europea''- (purified from PCR run on 04.09)
 +
 
Primers: 18a & 18b
Primers: 18a & 18b
-
Two versions: one with 5% DMSO and one with 3% DMSO
 
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(x5)
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Two versions, one with 5% DMSO and one with 3% DMSO and two programs: Touchdown (62C -> 55) and a single annealing temperature of 59C.
 +
 
[[Team:DTU-Denmark/Methods/PCR-mix|PCR reaction master mix]]
[[Team:DTU-Denmark/Methods/PCR-mix|PCR reaction master mix]]
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[[Team:DTU-Denmark/Methods/Determining_concentration_of_nitrogen_compounds/Experiment_2|Experiment 2]]
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{| class="wikitable" style="text-align: right"
{| class="wikitable" style="text-align: right"
! compound !! volume (uL)
! compound !! volume (uL)
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*Add 49 ul of the master mix, and
*Add 49 ul of the master mix, and
*Add 1 ul DMSO (for the 5%) or 1 ul of MQ (for the 3%)
*Add 1 ul DMSO (for the 5%) or 1 ul of MQ (for the 3%)
 +
 +
===User reaction===
 +
 +
was performed with HAO User fragment that was purified yesterday (but had low concentration) and pZA21::ara (promoter from the Biobrick K808000) and pZA21 with a tight arabinose promoter (from our SPL). Also Nir 1 User and Nir 2 User were ligated into the pZA21 ara tight for Nir. Negative controls were made for all three backbones.
==Results==
==Results==
<hr/>
<hr/>
-
==Conclusion==
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-
<hr/>
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===Gel 1===
 +
Gel of yesterday's PCR was empty (we lost the picture though...)
 +
 
 +
PCR was redone.
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 +
===Gel 2===
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For the PCR results of today.
 +
 
 +
* 2-log ladder
 +
* HAO User on 59C, 3% DMSO
 +
* HAO User on 59C, 5% DMSO
 +
* 2-log ladder
 +
 
 +
[[File:2013-09-06 HAO USER 59.jpg|600px]]
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 +
===Gel 3===
 +
 
 +
* 2-log ladder
 +
* HAO User on touchdown, 3% DMSO
 +
* HAO User on touchdown, 5% DMSO
 +
* 2-log ladder
 +
 
 +
[[File:2013-09-06 HAO USER td.jpg|600px]]
 +
 

Latest revision as of 10:55, 4 October 2013

6 September 2013

Navigate to the Previous or the Next Entry

Contents

Lab 208

Main purpose

  • Add USER endings to HAO gene extracted from Nitrosomonas europaea

Who was in the lab

Henrike

Procedure

Repeat HAO USER PCR from yesterday with same conditions and programs

Template: HAO extraction fragment -isolated from Nitrosomonas europea- (purified from PCR run on 04.09)

Primers: 18a & 18b

Two versions, one with 5% DMSO and one with 3% DMSO and two programs: Touchdown (62C -> 55) and a single annealing temperature of 59C.

PCR reaction master mix

compound volume (uL)
dNTPs 1
HF 10
polymerase x7 0.5
FW 3
RV 3
template 1
MQ 29
DMSO 1.5
  • Add 49 ul of the master mix, and
  • Add 1 ul DMSO (for the 5%) or 1 ul of MQ (for the 3%)

User reaction

was performed with HAO User fragment that was purified yesterday (but had low concentration) and pZA21::ara (promoter from the Biobrick K808000) and pZA21 with a tight arabinose promoter (from our SPL). Also Nir 1 User and Nir 2 User were ligated into the pZA21 ara tight for Nir. Negative controls were made for all three backbones.

Results

Gel 1

Gel of yesterday's PCR was empty (we lost the picture though...)

PCR was redone.

Gel 2

For the PCR results of today.

  • 2-log ladder
  • HAO User on 59C, 3% DMSO
  • HAO User on 59C, 5% DMSO
  • 2-log ladder

2013-09-06 HAO USER 59.jpg

Gel 3

  • 2-log ladder
  • HAO User on touchdown, 3% DMSO
  • HAO User on touchdown, 5% DMSO
  • 2-log ladder

2013-09-06 HAO USER td.jpg


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