Plasmid controls

To provide controls for the PCR and for initial optimisation of the assays, regions of the PLOD1 gene that included the region of interest were synthesized commercially and inserted into a pEX-A128 plasmid vector (Eurofins Genomics). These plasmids were prepared in standard DH5-alpha E-coli using a Qiagen mini-prep kit. This method produced control plasmids that were approximately 60 ng/μl in concentration. Individually the plasmids were used as the homozygous controls, and when mixed in equal concentrations together they acted as the heterozygous control.