Team:DTU-Denmark/Notebook/12 July 2013

From 2013.igem.org

(Difference between revisions)
(→Main purpose)
(→Amplification of cytochromes, AMO, HAO and Nir genes)
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*7, 8, 9 -> HAO
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*10, 11, 12 -> Nir
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[https://2013.igem.org/Team:DTU-Denmark/Methods/PCR| PCR] reaction mix was done according to standard procedure.Primers and tamplates used for samples are as follows:
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*1, 2, 3 -> cytochromes, primers 16a, 16b, template CYC isolated by colony PCR from ''Nitrosomonas europea''
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*4, 5, 6 -> AMO, primers 17a, 17b, template AMO isolated by colony PCR from ''N. europea''
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*7, 8, 9 -> HAO, primers 18a, 18b, template HAO isolated by colony PCR from ''N. europea''
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*10, 11, 12 -> Nir, primers 15a, 15b, template - 2uL of liquid culture of "Pseudosomonas"
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PCR programs based on [https://2013.igem.org/Team:DTU-Denmark/Methods/PCR| standard program] with differences in annealing temperature and elongation time as follows:
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*1, 2, 3 -> cytochromes - 57°C, 1:30 min
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*4, 5, 6 -> AMO - 54°C, 3 min
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*7, 8, 9 -> HAO - 59°C, 9 min
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*10, 11, 12 -> Nir - 59°C, 9 min

Revision as of 15:45, 12 July 2013

Contents

208

Main purpose

  • miniprep of biobrick transformants from 10-07-2013
  • PCR with USER primers to amplify cytochromes, AMO, HAO and Nir genes
  • gel analysis of PCR products from Nir operon, AraBAD promoter and RFP
  • USER reaction of RFP and pZA21 (with native promoter) and transformation of E. coli cells

Who was in the lab

Henrike, Julia, Kristian, Jakob, Gosia

Procedure

Amplification of cytochromes, AMO, HAO and Nir genes

Each PCR reaction was performed in triplicate.

Samples are named:

  • 1, 2, 3 -> cytochromes
  • 4, 5, 6 -> AMO
  • 7, 8, 9 -> HAO
  • 10, 11, 12 -> Nir

PCR reaction mix was done according to standard procedure.Primers and tamplates used for samples are as follows:

  • 1, 2, 3 -> cytochromes, primers 16a, 16b, template CYC isolated by colony PCR from Nitrosomonas europea
  • 4, 5, 6 -> AMO, primers 17a, 17b, template AMO isolated by colony PCR from N. europea
  • 7, 8, 9 -> HAO, primers 18a, 18b, template HAO isolated by colony PCR from N. europea
  • 10, 11, 12 -> Nir, primers 15a, 15b, template - 2uL of liquid culture of "Pseudosomonas"

PCR programs based on standard program with differences in annealing temperature and elongation time as follows:

  • 1, 2, 3 -> cytochromes - 57°C, 1:30 min
  • 4, 5, 6 -> AMO - 54°C, 3 min
  • 7, 8, 9 -> HAO - 59°C, 9 min
  • 10, 11, 12 -> Nir - 59°C, 9 min
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