16S rRNA of Espeletia Microbiome: Young leaves - Endophyte Community
The V5-V6 hypervariable region of the 16S rRNA gene of Bacteria and Archaea was amplified with primer 799F (5-AACMGGATTAGATACCCKG-3), which minimizes contamination from chloroplast DNA and amplifies a mitochondrial product that is larger and thus easier to separate and differentiate from the microbial amplified products (Chelius and Triplett 2001), and the reverse primer 1050R (5-AGYTGDCGACRRCCRTGCA-3) (Bohorquez et al 2012). DNA concentration was adjusted, as previously reported (Bodenhausen et al 2013) and used in 25uL PCR reactions containing: DNA (10ng for endophytic fraction or 1ng for epiphytic fraction), 2.5ľL 10X AccuBuffer (600mM Tris-HCl, 60mM (NH4)2SO4, 100mM KCl, 20mM MgSO4, pH 8.3), 2ľL 10mM dNTP Mix, 0.5uM of each primer and 5 Units of ACCUZYME DNA Polymerase (Bioline USA Inc.; Taunton, MA; USA). Cycling conditions were 94şC for 2 min, followed by 30 cycles of 94şC for 30sec, 55şC for 30sec, 72şC for 45sec; and a final extension of 72şC for 10min. PCR products were separated on a 3% agarose gel and purified based on amplicon size difference using the QIAquick gel extraction kit (QIAGEN, Valencia, CA; USA), when necessary. The remaining samples were purified using the QIAquick PCR Purification Kit (QIAGEN, Valencia, CA; USA). DNA was quantified using QubitŽ 2.0 Fluorometer (Life Technologies Corporation, Carlsbad, CA; USA) and amplicons were sequenced on an Illumina MiSeq machine with a Paired-End protocol (PE-250) (Molecular Research MR DNA, Shallowater, TX, USA).