454 sequencing analysis of insect-based products during cold storage
PCR amplification was performed on each DNA sample in a reaction volume of 40 ľl, containing 0.15 mM of each dNTP (Invitrogen, Merelbeke, Belgium), 0.5 ľM of each primer, one unit Titanium Taq DNA polymerase, 1 x Titanium Taq PCR buffer (Clontech Laboratories, Inc., Palo Alto, CA, USA), and 10 ng genomic DNA (measured using a Nanodrop instrument (Thermo Scientific Nanodrop Products Inc., Wilmington, DE, USA)). PCR conditions were as follows: initial denaturation at 94°C for 2 min followed by 35 cycles of denaturation at 94°C for 45 s, primer annealing at 59°C for 45 s, extension at 72°C for 45 s, and a final extension step at 72°C for 10 min. After resolving the amplicons by agarose gel electrophoresis, amplicons within the expected size range were excised and extracted/purified from the gel using the Qiaquick gel extraction kit (Qiagen, Hilden, Germany). Purified dsDNA amplicons were then quantified with the Qubit fluorometer with the high-sensitivity DNA reagent kit (Invitrogen, Carlsbad, CA, USA) and combined at equimolar concentrations (1.00 x 109 molecules/ml) of each amplicon.