The cycling conditions consisted of an initial denaturation at 98◦C for 3min, 25 cycles of: 98◦C for 10 s, 50◦C for 20 s, and 72◦C for 20 s, and a final extension at 72 ◦C for 10min. PCRs were performed with an Alpha cycler 1 (PCRmax, Staffordshire, UK) using an adaptation of the cycling conditions of Caporaso et al. The PCR protocol I followed is : The 10-20 ng of DNA was used as template in the PCR reaction (50µL), which contained 10µL HF buffer (Thermo Fisher Scientific), 1µL dNTP Mix (10mM Bioline, London, UK), 1U of Phusion Hot Start II DNA Polymerase (Thermo Fisher Scientific), 500 nM of each barcoded primer. I haven't came across the issue before and the negative control was fine (which exclude the contamination from water or materials). However, besides the target products, I found some bigger products (about 550 bp) appeared. My primers are supposed to target the V4 region and amplify the products around 330bp. I used Phusion Hot start II DNA polymerases kit. I am trying to use PCR to amplify my samples with barcoded primers for 16s sequencing.
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