Begin by linearizing the vector. Prepare the reaction mixture containing specific restriction enzymes at room temperature. Use a gel extraction kit to purify the linearized vectors, then incubate a 37 degrees Celsius for 60 minutes.
Then, assemble the exon R seamless cloning and assembly reaction to sub-clone the PRRSV gene. Adjust the total reaction volume to 10 microliters with sterilized deionized water. Then, thoroughly mix.
Incubate the mixture in a thermocycler for 15 to 60 minutes at 50 degrees Celsius. Afterwards, store the samples on ice. After transforming the vector into competent cells, pick eight colonies into 20 microliters of LB medium containing kanamycin.
Set up a colony PCR reaction to analyze the transformants. A full length PRRSV over-expression vector was obtained by introducing PRRSV fragment one into the pVAX1 vector, creating pVAX1-F1. Successive rounds of recombination using specific restriction enzymes resulted in incorporation of fragments two through five, visualized by an increase in plasmid size.
