100 bp dna ladder marker

Four oligonucleotide primer sets (synthesized by Macrogen Inc. Korea) derived from the mitochondrial DNA cyt b gene sequences for the amplification of chicken, beef, and pork, as designed by Matsunaga et al. [15] , were used for the PCR amplification. The primer sequences are as given in Table 2. Simplex PCR amplification was accomplished in a 25 µl total volume containing 0.625U GoTaq Flexi DNA Polymerase (Promega, Madison, USA), 5 µl of 5X GoTaq Flexi Buffer, 200 µM each of dNTP, 1.5 mM MgCl2, 0.4 µM primers and 1 µl (30 ng/µl) of total DNA in thermal cycler GeneAmp® PCR System 9700 (Applied Biosystems, Foster City, CA) and the cycling parameters were initial denaturation at 94 o C for 3 min following 35 cycles of denaturation at 94 o C for 30s, annealing at 60 o C for 30s and elongation at 72 o C for 30s and final elongation at 72 o C for 3 min.
Five µl of the PCR products from each amplified product was electrophoresed along with a 100-bp DNA ladder marker (Promega) to confirm the targeted PCR amplification. The electrophoresis was performed on 2% agarose gel containing ethidium bromide (0.5 µg/ml) at constant 80 V for 60 min in 1X Tris-acetate EDTA (TAE) buffer. The bands in the gel were then visualized using PhotoDoc-It Imaging System (UVP)

Genomic DNA extraction was performed using a synthetic resin (Instagene Bio-Rad Matrix), following the protocol described by Capece et al. (2011) (link). The presumptive identification of the yeasts obtained from traditional Turkish cheeses was performed by PCR amplification of the internal transcribed spacers between the 18S and 26S rDNA genes (ITS1-5.8S-ITS2) and subsequent restriction analysis, as reported by Esteve-Zarzoso et al. (1999) . PCR products were digested without further purification with restriction enzymes HaeIII and HinfI (Promega, USA). Restricted fragments were analyzed by electrophoresis on 2% agarose gels, and 1.0X TBE buffer, stained with SyberSafe DNA gel stain (Invitrogen, USA) and the obtained profiles were visualized and photographed with Gel Doc TM XR+ (Bio-Rad, USA) under UV light. A 100-bp DNA ladder marker (Promega, USA) served as the standard size. Isolates showing the same restriction pattern were grouped, and one or two representative yeasts for each group were delivered to Eurofins Genomics Srl (Vimodrone, Italy) for sequencing. The sequences received were compared with those deposited in the GenBank DNA database (National Center for Biotechnology Information, 2015) using the basic BLAST search tools (Altschul et al., 1997) (link).