title: 16S of Lake Macleod Bacterioplankton
design: Samples were PCR amplified with universal 454 bacterial primers (27F/519R with barcode on the forward primer) targeting the 16S rRNA gene V1-V3 variable region. PCR included 30 cycles using the HotStarTaq Plus Master Mix Kit (Qiagen, USA): 94°C for 3 minutes, followed by 28 cycles of 94°C for 30 seconds, 53°C for 40 seconds and 72°C for 1 minute, then 72°C for 5 minutes. PCR products were pooled and purified then DNA libraries were prepared following the Illumina TruSeq DNA library preparation protocol. Sequencing was performed on a MiSeq following the manufacturer’s guidelines. The r1 and r2 files for amplicons 460 greater than 300bp and less than 570bp were joined at the Molecular Research Laboratories (Shallowater, TX, USA).