roots_RM_endophyte
Primer 799F (5'-AACMGGATTAGATACCCKG-3'), which minimizes contamination from plastid DNA (Chelius and Triplett, 2001) and a primer designed for this study, 1193r (5'-ACGTCATCCCCACCTTCC-3'), were used to amplify V5, V6 and V7 of the 16S rRNA gene. The forward primer was fused to the 454 Life Sciences primer B and the reverse primer was fused to the adapter A and a barcode in order to sequence the hypervariable regions V6 and V7.  Each 25 µl PCR reaction contained 10 ng (for the endophytic fraction) or 1 ng (for the epiphytic fraction) of DNA, Mg2+ free PCR buffer (TaKaRa), 3mM MgCl2 (TaKaRa), 200 µM dNTP, 200 nM forward primer, 200 nM reverse primer, 12.5 µg ultrapure BSA (Ambion), and 1 unit Ex Taq HotStart polymerase (TaKaRa). Cycling conditions were 94ºC for 2 min, followed by 25 cycles of 94ºC for 30 sec, 55ºC for 30 sec, 72ºC for 1 min, with a final extension of 72ºC for 10 min. All samples were amplified in quadruplicates, which were combined before purification. Primer 799f and 1193r amplify a mitochondrial product of about 800 bp and a bacterial product of about 500 bp. We isolated the bacterial product by separating the PCR products on a 3% low melt agarose gel (2% agarose for root samples) and excising a band of agarose with size 400 bp to 700 bp. DNA was extracted from the gel using the QIAquick gel extraction kit (Qiagen). After purification, DNA was quantified using the PicoGreen assay (Invitrogen) and the quality was checked using a Bioanalyzer (Agilent). DNA concentration was adjusted to 1 ng/µl. The amplicon libraries were prepared by pooling 10 ng of each PCR.