Once a suitable genomic fragment is chosen (Step 1), the exons and introns of the fragment can be inserted and amplified, along with the flanking constitutive exons and introns of the original gene, by PCR. The primers for PCR can be chosen so that they leave "sticky ends" at 3' sense and anti-sense strands (Step 2). These "sticky-ends" can be easily incorporated into a TOPO Vector by ligation into a commercially available source which has ligase already attached to it at the sight of incorporation  (Step 3). The subsequent TOPO Vectors can be transfected into E.coli cells (Step 4). After incubation, total RNA can be extracted from the bacterial colonies and analyzed using RT-PCR to quantify ratios of exon inclusion/exclusion (step 5). The minigene can be transfected into different cell types with various splicing factors to test trans-acting elements (Step 6). The expressed genes or the proteins they encode can be analyzed to evaluate splicing components and their effects via a variety of methods including hybridization or size-exclusion chromatography.