Extract n amp pcr ready mix

Total genomic DNA was extracted from silica-dried leaves using Extract-N-Amp™ Plant PCR Kits (SIGMA - Aldrich, St. Louis, MO, USA) abiding by the supplier’s instructions. Fifteen nuclear SSR markers previously selected for studying the genetic diversity of Prunus species in the Mediterranean region were used for this study: UDP96–001, UDP96–018, UDP96–003, UDP97–401, UDP98–408, UDP98–409, pchgms1, pchgms3, BPPCT017, BPPCT001, BPPCT007, BPPCT025, BPPCT036, CPSCT018, CPDCT045 [34 (link)–38 (link)]. We amplified these 15 SSR markers into three multiplexed PCRs, using one of the FAM, HEX or NED fluorophore-labeled primers (PE Applied Biosystems, Warrington, UK). Multiplexed PCRs were carried out with the Extract-N-Amp PCR Ready Mix (SIGMA - Aldrich, St. Louis, MO, USA) in a final volume of 10 μl, containing 5 μl of a SIGMA Master Mix 2X, 0.4 μl of primer mix at 5 μM, 1.6 μl of ultrapure water and 1 μl of template dNTPs. 3 μl of PCR product was mixed with 15 μl of formamide and 0.2 μl of Genescan™ 500 LIZ size standard (Applied Biosystems, Foster City, USA), and GeneScan was performed with the ABI PRISM 3130 XL 16 capillary-sequencer (Applied Biosystems, Foster City, USA).