This set of experiments focuses on the amplification of two PCR products: one for claudin-2 and one for claudin-12. The claudins are components of tight junctions found between intestinal cells and are involved in creating a permeability barrier so that substances cannot pass from the lumen of the intestine to the blood.
calculate and estimate optimal annealing temperature for primers of claudin-2 and claudin-12 DNA sequences,
Table 1 shows the primers used in this exercise to amplify intestinal cDNA for claudin-2 and claudin-12. Students can calculate the optimal annealing temperature on the basis of primer compositions and design an experiment to test different temperature ranges in order to determine the optimal annealing temperature.
In the second module, cDNA is used in PCR to amplify cDNA for claudin-2 and claudin-12 at varying annealing temperatures.
The cDNA generated from the RNA is used in standard PCR with Taq polymerase and gene-specific primers for claudin-2 and claudin-12. Primer sequences for claudin-2 and claudin-12 are shown in Table 1, with details concerning composition and annealing temperatures.
Separating the PCR products through an agarose gel and staining with ethidium bromide (Figure 2) shows one clear band at the expected length for each primer set: 692 bp for claudin-2 and 604 bp for claudin-12. Each set of primers shows an enhanced PCR product just below the calculated annealing temperature (60[degrees]C for claudin-2 and 67[degrees]C for claudin-12).