Team:DTU-Denmark/Notebook/9 September 2013

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(→Colony PCR to verify inserts HAO and Nir in pZA21::ara and pZA21::ara tight)
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===Colony PCR to verify inserts HAO and Nir in pZA21::ara and pZA21::ara tight===
===Colony PCR to verify inserts HAO and Nir in pZA21::ara and pZA21::ara tight===
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Colony PCR on the Nir transformants, testing for NirH (primers 45a, 45b, annealing 59C), NirM+S (primers 44, 11a annealing 64C) and NirN (primers 43, 11b annealing 68C).
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program:
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{| class="wikitable" style="text-align: right"
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! temperature !! time !! cycles
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|-
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|  98C || 10:00 || -
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|-
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|  98C || 0:10 || 36
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|-
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|  59C/64C/68C || 0:30 || 36
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|-
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|  72C || 2:00 || 36
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|-
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|  72C || 5:00 || -
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|-
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|  10C || hold || -
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|-
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|}
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Colony PCR to verify HAO insert.
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Primers: 28a, 28b
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temperature: 59C
==Results==
==Results==

Revision as of 12:02, 11 September 2013

9 September

Contents

lab 208

Main purpose

Who was in the lab

Julia, Kristian, Henrike

Procedure

Colony PCR to verify inserts HAO and Nir in pZA21::ara and pZA21::ara tight

Colony PCR on the Nir transformants, testing for NirH (primers 45a, 45b, annealing 59C), NirM+S (primers 44, 11a annealing 64C) and NirN (primers 43, 11b annealing 68C).

program:

temperature time cycles
98C 10:00 -
98C 0:10 36
59C/64C/68C 0:30 36
72C 2:00 36
72C 5:00 -
10C hold -

Colony PCR to verify HAO insert.

Primers: 28a, 28b

temperature: 59C

Results

Transfomation

Transformation on last friday gave the following results:

  • HAO in pZA21::ara (from Biobrick K808000) - 6 colonies (3 orange and small, 3 normal color and very big)
  • negative HAO in pZA21::ara (from Biobrick K808000) - no colonies
  • HAO in pZA21::ara tight - a lot of colonies, some very big
  • negative HAO in pZA21::ara tight - a lot of small colonies
  • Nir in pZA21::ara tight - 8 colonies
  • negative Nir in pZA21::ara tight - no colonies


Gel

Gel on the results of the first colony PCR on the Nir transformants, testing for NirH, NirM+S and NirN

  • 1 kb ladder
  • colony 1, NirH
  • colony 1, Nir M+S
  • colony 1, NirN
  • colony 2, NirH
  • colony 2, Nir M+S
  • colony 2, NirN
  • colony 3, NirH
  • colony 3, Nir M+S
  • colony 3, NirN
  • colony 4, NirH
  • colony 4, Nir M+S
  • colony 4, NirN
  • 1 kb ladder

2013-09-09 nir col.jpg

The gel picture is inconclusive. We will do another colony PCR using the primers designed for sequencing.

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