The amount of fragmented DNA actually used in the PCR amplification step is typically in the low-attomole range, so the theoretical amount of starting DNA needed for DS is extremely low. However, when feasible, it is most convenient to start with excess amounts of DNA to allow for easy quantification of the DNA throughout the initial sample preparation steps and to account for expected sample loss at the enzymatic and purification steps. Typically, 1–3 μg of DNA is required for targeted DNA capture and 100–300 ng of DNA for smaller genomes, such as purified plasmid or mtDNA, that do not require targeted capture. When performing DNA isolation, it is imperative to avoid any manipulations that could result in melting of the two DNA strands, such as heating above 80 °C, the use of chaotropic agents such as urea or overdrying the DNA during ethanol precipitations.
