Phusion high fidelity pcr master mix

RNA extractions were performed immediately after lysis by using TRIzol solution (Ambion), followed by chloroform phase separation and isopropanol/ethanol RNA precipitation. After a first quantification by Qubit 2.0 fluorometer using Qubit RNA HS Assay Kit (Life Technologies, Carlsbad, CA), 10 μg total RNA aliquots were treated with Turbo DNA-free kit (Ambion), according to the manufacturer’s instructions. After 30 minutes of incubation and final addition of the inactivation reagent, the supernatant was recovered and the Qubit 2.0 fluorometer was used to quantify the RNA.
Capillary electrophoresis on an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA) was used to analyze approximately 3 ng of each RNA, using the RNA 6000 Pico LabChip kit. All RNA samples showed RIN values between 8 and 9.7. Assessment of the efficiency of DNAse treatment by PCR on 10 ng total RNA using strain-specific primers completed the validation of the samples. Phusion High Fidelity PCR Master Mix (Life Technologies) was used to run reactions which included a positive (fresh colony lysate in water) and a negative (nuclease-free water) amplification control. Equimolar amounts of total RNA from the four species were pooled to form the MIX sample. This was the starting material for all the synthetic metatranscriptome experiments.

Basic cloning steps were performed using the following tools and appropriate protocols: for DNA purification, a QIAquick PCR purification kit (QIAGEN) was used. Plasmid minipreps were performed using the GeneJET Plasmid Miniprep kit (Life Technologies). All PCR reactions for plasmid construction were performed using the Phusion High-Fidelity PCR Master Mix (Life Technologies), and all diagnostic PCR reactions were performed using DreamTaq DNA Polymerase (Life Technologies). Oligonucleotides were synthesized by Sigma-Aldrich and Eurofins Genomics. Oligonucleotides were phosphorylated by T4 polynucleotide kinase (NEB). DNA was sequenced by GATC Biotech and Eurofins Genomics.

Genomic DNA was extracted from cultures harvested in the exponential growth phase using the DNeasy Plant mini kit (Qiagen, Netherlands) according to the manufacturer’s instructions. Partial 28 S ribosomal DNA (rDNA) genes were PCR amplified using the forward primer Leuk2F (5′-acccgctgaacttaagcatatcact-3′) and the reverse primer Leuk_34r (5′-gcatcgccagttctgcttacc-3′). PCRs were performed in a total reaction volume of 25 μL using the Phusion high-fidelity PCR master mix with GC buffer (Finnzymes, Finland). The PCR protocol employed was as follows: 30 s initial denaturation at 98 °C, followed by 30 cycles of 10 s at 98 °C, 30 s annealing at 55 °C and 1 min extension at 72 °C. A final 5-min extension step at 72 °C was conducted to complete the amplification. Amplification products were controlled by electrophoresis on a 1% agarose gel. PCR products were sequenced directly on an ABI PRISM 3100 xl DNA autosequencer (Perkin-Elmer Inc., USA) using the ABI PRISM BigDye Terminator Cycle Sequencing Kit (Perkin-Elmer). Alignments were generated using MUSCLE implemented in MEGA v6.0641 (link) with subsequent manual verification.