Results

PCR of nadD, nadE and PncB

The three aforementioned genes were PCR amplified initially without the iGEM prefix and suffix, which would enable the correct hybridisation of the primer to the E.coli genomic DNA to amplify the genes. The forward and reverse primer sequences for these genes are: (see table)

These primers were successful in amplifying these genes and was confirmed through gel electrophoresis. However as PncB has a Pst1 site which is incompatible with the biobrick system, internal primers were used which had a single base change (from a G to a C) to remove the restriction site. The Forward Internal and the Reverse Exterior primer were used for one PCR reaction and in another it was the Reverse Internal primer and the Forward Exterior primer. This resulted in two PCR products overlapping at the removed restriction site. This was confirmed by a gel. The forward and reverse exterior primers were then used to amplify these products into one PncB amplification and thus having a PncB without a restriction site. This was confirmed by digesting this PncB with Pst1 and resulted in one band, as can be seen in figure 1. The PCR product that was successful was purified and had concentrations of 8.2ng/µl on average. (see figure 1)

Discussion

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