Team:DTU-Denmark/Notebook/9 September 2013

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=lab 208=
=lab 208=
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Revision as of 22:01, 16 September 2013

9 September

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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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