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The method of PCR was used for the rapid and specific amplification of DNA fragments of interest here pUC18 vectors with the fdx4 insert Besides restriction sites within the PCR primer sequences were built in for later cloning steps too The standard PCR protocol and the components for the PCR reaction which were used are shown in Table A1 Annealing temperature and elongation time were adjusted based on primer characteristics and the product size respectively PCR products were analyzed and separated by agarose gel electrophoresis These separated DNA fragments were excised and purified using a PCR purification kit 2 2 Agarose gel electrophoresis Agarose gel electrophoresis was performed to separate DNA fragments according to their size and therefore used to confirm each step of the cloning process Agarose gel was prepared by adding appropriate amount of agarose into TAE buffer samples were mixed with 6x loading dye including SYBR Green and the DNA fragments were separated in an electric field 80 120 V The agarose concentration of the gels was set to 1 0 according to the size of DNA fragments to be analyzed A 1 kbp DNA marker was applied as a size standard Upon finishing the gels were documented using a blue light transilluminator and a camera to take a photo If necessary DNA bands of interest were excised from the gel and the DNA was subsequently extracted using a QIAquick PCR Purification Kit according to the manufacturer s instructions 2 3
