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From 2013.igem.org

Contents 1 208 1.1 Main purpose 1.2 Who was in the lab 1.3 Procedure 1.3.1 Plasmid isolation of biobricks from transformants made on 10-07-2013 1.3.2 Amplification of cytochromes, AMO, HAO and Nir genes 1.3.3 Gel analysis of PCR products from Nir operon, AraBAD promoter and RFP

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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

Plasmid isolation of biobricks from transformants made on 10-07-2013

Isolation was performed according to protocol given in the kit PowerPrep HP Plasmid Miniprep Kit

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

Gel analysis of PCR products from Nir operon, AraBAD promoter and RFP