To begin, open the software and select the option to create a new DNA file. Prepare the PRRSV gene sequence from NCBI and click Okay to generate the sequence files. Analyze the sequence and mark the fragment junctions.
Identify all the junctions before designing the specific sequence primers for the fragments. Then click on Primers and choose Add Primer. Paste the specific sequence and add overlap sequences of the vector to the first five prime nucleotide of the primer.
Name the forward primer and click on Add Primer to Template to incorporate the designed primer into the template. Then mark the fragment junctions and design the reverse specific sequence primer of the fragments. Again, navigate to Primers and select Add Primer.
Input the specific sequence. Add the restriction site to the first five prime nucleotide. Also, include 20 to 40 base pairs of vector overhang sequences to the first five prime nucleotide of the restriction site.
Name the reverse primer and select Add Primer to Template. Next, set up six individual PCR reactions using the six fragments and designed primer pairs. Run the PCR following a three-step protocol as shown here.
After PCR completion, pipette one microliter of six xDNA loading buffer with five microliters of the PCR product. Mix and centrifuge briefly. Analyze the samples using 1%agarose gel electrophoresis.
To construct a full length gene, insert fragments of PRRSV were amplified using six PCR reactions. The sizes of the fragments were confirmed by agarose gel electrophoresis.
