Phire hot start dna polymerase

As part of the sample analysis, bacterial species composition was studied through amplicon sequencing of ribosomal DNA from these soil samples. Amplicon libraries were prepared by running a PCR with fusion primers optimized for 454 sequencing. Fusion primers for bacterial 16S rDNA V3-V4 region were designed using the forward primer B341F [‘CCTACGGGNGGCWGCAG’] and 454 (Lib-A) adapter-A and the reverse primer B805R [‘GACTACHVGGGTATCTAATCC’] preceded by 454 (Lib-A) adapter-B with a MID.^{31 (link)} All amplicon libraries were prepared using reagents based on Phire Hot Start DNA Polymerase (Thermo Fisher Scientific Inc., Waltham, MA, USA). The pre-PCR mix was prepared in the same proportion for each sample totaling to 25 µl (5 µl-5× Buffer; 0.5 µl-10 mM dNTPs; 1 µl-10 µM forward primer; 2 µl-5 µM reverse primer; 0.5 µl-DNA polymerase; 0.5 µl-bovine serum albumin; 2.5 µl-Template DNA; 13 µl-MilliQ water). The PCR conditions for 16S rDNA amplification were as follows: initial denaturation step at 98°C for 30 s; 27 cycles of denaturation at 98°C for 5 s, annealing at 56°C for 5 s, extension at 72°C for 10 s; final extension at 72°C for 60 s. Three 25 µl PCR reactions were run separately for each sample and pooled together. These PCR amplicons were purified with QIAquick PCR purification kit (Qiagen), and the quantity was measured using Quant-it Pico Green kit (Invitrogen).

The bacterial strains and plasmids used in this study are described in Table 2. Oligonucleotides were purchased from Integrated DNA Technologies and are listed in Table 3. Genomic DNA was isolated using the Qiagen 100/G genomic tip (Qiagen). Weakening of the staphylococcal cell wall required the addition of 100 µg of lysostaphin (Ambi) into the lysis buffer and incubation at 37°C for 30 min. Plasmids and PCR products were isolated using the Wizard plus kits (Promega), with T4 DNA ligase also purchased from Promega. Plasmids were isolated from staphylococci as described previously (3 (link)). Restriction enzymes, T4 DNA polymerase, and Phusion DNA polymerase were purchased from New England Biolabs. Phire Hotstart DNA polymerase was purchased from Thermofisher. Sanger sequencing was supplied by Eurofins. Routine manipulation of S. aureus and E. coli was performed as described by Monk et al. (3 (link)). X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; Melford) was used at 50 µg/ml in E. coli and 100 µg/ml in S. aureus. Antibiotics were purchased from Sigma Aldrich and used at the following concentrations: carbenicillin (Car), 100 µg/ml; chloramphenicol (Cm), 10 µg/ml; and kanamycin (Kan), 50 µg/ml (E. coli) and 100 µg/ml (S. aureus).