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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 Amplification of cytochromes, AMO, HAO and Nir genes

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

===Plasmid isolation of biobricks from transformants made on 11-07-2013

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