IPCR within a Circular Construct

A quality target genomic DNA extract should be obtained, and 500 ng digested with an appropriate restriction enzyme in a 30 pL volume and adequate digestion should be confirmed by agarose gel electrophoresis. The resulting fragment should contain a region of known sequence flanked by the unknown target. The enzyme is then inactivated, DNA-precipitated, and diluted (approximately 1:5) prior to ligation in a media of ligase buffer, water, and ligase to optimize conditions for intramolecular ligation. Following incubation according to the manufacturer's instructions (approx. 1-12 hr at room temperature), complete ligation with production of a circular construct can be verified by Southern blotting using the known sequence as a probe.

A titration series (5-10 pL) of the circular construct should be used for the PCR reaction in a final volume of 50 ml to optimize the chances of target DNA amplification. Primers should be designed based on the known sequence and PCR conditions to include a high annealing temperature to prevent any nonspecific priming, a long extension time (2 min), and up to 40 cycles to maximize the chances of amplification of rare templates.

A nested PCR maybe performed to increase the specificity of the reaction (Fig. 1). In this case, following the first-round PCR and analysis of the product by agarose gel electrophoresis, another titration series of the first-round product (up to 15 pL) should be added to a second-round PCR reaction containing primers located inside the region previously amplified. Annealing temperatures should be increased with an extension time of 1 min (approx.). Final products should be analyzed by electrophoresis.

In general, the objective of inverse PCR is to elucidate the sequence of the unknown region, and so the inclusion of a proofreading enzyme (e.g., PfU) in the PCR reactions will reduce errors that may be introduced during amplification. Products obtained after PCR are then cloned into a vector for subsequent DNA sequencing.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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