Dig labeled size markers 7 and 8

1 µg of gDNA was digested with HaeIII (37 °C for 12 h) and the enzyme subsequently inactivated at 80 °C for 20 min. Serial dilutions were made in water to a final concentration of 90 pg/µl and 1 µl (approx. 30 genome equivalents, g.e.) was used for PCR amplification using a non-FAM-labeled version of the XDP SVA hexamer primers with the small pool-PCR conditions outlined in Additional File 1: Table S1. For each sample, PCR amplifications of 36 replicates of 90 pg gDNA and 8 DNA-negative PCR controls were carried out in a 25 µl reaction volume with buffer and dNTPs provided with the PrimeSTAR GXL polymerase (Takara) according to the manufacturer’s protocol. 10 µl of each PCR product were run in 2% agarose gels alongside digoxigenin (DIG)-labeled size markers VII and VIII (Roche), for 16 h at 50 V then transferred to a positively charged nylon membrane (Roche) by common squash-blotting technique [25 (link), 46 (link)]. The membrane was hybridized with 5 pmol/ml of a 5’ DIG-labeled (AGAGGG)₁₀ probe (Sigma) in DIG Easy Hybridization Solution (Sigma) overnight at 45 °C and then washed twice each with 2 X SSC, 0.1% SDS at room temperature for 5 min, 0.1 X SSC, 0.1% SDS at 68 °C for 20 min, and 0.1 X SSC, 0.5% SDS at 68 °C for 20 min. DIG detection was carried out using the DIG Luminescent Detection system (Sigma) with CPSD substrate according to the manufacturer’s instructions.