Simplex PCR Assay for Sex Determination in Mice

BENCHMARKS dsDNA was removed from the super-natant (lane 4), which was discarded. The bead-bound dsDNA was denatured in alkaline solution (0.1 M NaOH and 1 mM EDTA) at room temperature for 15 min, and the ssDNA without biotin groups was released in the alkaline solution (lane 5). After neutralization, the ssDNA solution was combined with five volumes of Buffer PB from the MiniElute™ PCR Purification Kit passed through MiniElute columns. The ssDNA was then eluted from the columns with a small volume of 10 mM Tris-HCl, pH 7.4 (lane 6). The recovery yield was 85% for this 149-nucleotide ssDNA. The whole procedure could be easily performed in 1 h either manually or by an automation workstation) with a robotic liquid handler, vacuum and manifold, washing unit, and a magnetic plate. Compared to the original protocols (1–3) or other standard methods involving gel purification and ethanol precipitation (4,5), this renovated protocol saves time and effort and is cost-effective (by a dramatic reduction in the use of expensive SA-coated superparamagnetic beads), although additional materials are used. The recovery yield of this protocol is a little lower than that (approximately 95%) of the original ones (1–3) because the binding affinity of silica membrane is weak for short DNA (approximately 75% for an 88-nucleotide ssDNA; data not shown) while alcohol precipitation can effectively recover short nucleic acids from solution. When short ssDNA (60–75 nucleotides) is to be prepared with high recovery yield, instead of using silica membrane, this protocol can be modified by using a molecular sieve column (Sephadex ® G-25; Amersham Biosci-ences, Piscataway, NJ, USA) to desalt. However, the volume of prior alkaline and acid solutions must be reduced to increase the ssDNA concentration.