Biostabii pcr enhancer

PCR amplification and amplicon sequencing were performed by LGC Genomics (Berlin, Germany). The PCRs included about 5 ng of DNA extract, 15 pmol of each forward primer ITS1F 5′-NNNNNNNNNNTCTTGGTCATTTAGAGGAAGTAA and reverse primer ITS2R 5′-NNNNNNNNNNTGCTGCGTTCTTC ATCGATGC in 20 uL volume of 1x MyTaq buffer containing 1.5 units MyTaq DNA polymerase (Bioline) and 2 μl of BioStabII PCR Enhancer (Sigma). For each sample, the forward and reverse primers had the same 10-nt barcode sequence. PCRs were carried out for 35 cycles using the following parameters: 2 min 96°C pre-denaturation; 96°C for 15 s, 50°C for 30 s, 70°C for 90 s. DNA concentration of amplicons of interest was determined by gel electrophoresis. About 20 ng amplicon DNA of each sample were pooled for up to 48 samples carrying different barcodes. The amplicon pools were purified with one volume AMPure XP beads (Agencourt) to remove primer dimers and other small mispriming products, followed by an additional purification on MinElute columns (Qiagen). About 100 ng of each purified amplicon pool DNA was used to construct Illumina libraries using the Ovation Rapid DR Multiplex System 1-96 (NuGEN). Illumina libraries were pooled and size selected by preparative gel electrophoresis. Sequencing was done on an Illumina MiSeq using V3 Chemistry (Illumina).

We used 5ng of DNA extract for amplification of the barcoding region of the cytochrome c oxidase subunit I gene (CO1). For PCR amplification, we used the MyTaq DNA Polymerase kit (Bioline, Luckenwalde–Germany). For each reaction, 1.5U of MyTaq were pre-mixed with 20μL MyTaq buffer containing 15pmol of the forward and reverse primer, 2μL BioStabII PCR Enhancer (Sigma Aldrich, St. Louis–United States) and 1μL DMSO. We used four different amplicons targeting the CO1 gene (Table 2). For PCR of each DNA extract sample, a unique 8 base-barcode tag was used in the forward and reverse primer. DNA concentrations of amplified amplicons were checked via agarose gel electrophoresis. Approximately 20ng of amplified PCR product of each sample were transferred into amplicon-pools of up to 48 parallel samples. Samples yielding a lower amplicon concentration were amplified for another 5 cycles in an additional PCR reaction.