Dream taqtm green pcr master mix

A list with the oligonucleotides used for amplification and/or sequencing is provided as Additional file 1. For PCR amplifications, the following kits were used: Dream TaqTM Green PCR Master Mix (Fermentas) and Maxime PCR Premix Kit (i-Taq; Intron Biotechnology). The conditions for the PCRs were similar in all cases with slight differences depending on the fragment size and primers melting temperatures (see Additional file 1 for further details). Average amplification conditions were: 95 °C for 5 min followed by 30 cycles of 95 °C for 30 s, 54–62 °C for 30 s and 72 °C for 90 s followed by 72 °C for 10 min. PCR products were gel-purified and cloned into the pCR2.1 vector (Invitrogen). Plasmid DNA was prepared with the AccuPrep Plasmid Mini Extraction Kit (Bioneer).
The “Sanger” sequences were determined using the Big Dye Terminators v3.1 kit (Applied Biosystem, California, USA) by automatic sequencing at the Servicio de Genómica (Parque Científico de Madrid, Universidad Autónoma de Madrid). For sequencing regions with high G + C content the dGTPBigDye Terminator v3.0 Cycle Sequencing Ready Reaction Mix was also used. Sequences were carried out using the ABI Prism 3730 y 3730XL systems.

Total genomic DNA of the voucher specimens was extracted using the Sangon Fungus Genomic DNA Extraction kit (Sangon Biotech Co. Ltd., Shanghai, China), according to the manufacturer’s instructions. Primer pairs ITS5/ITS4 [14 (link)], LR0R/LR5 [15 (link)], and tef1F/tef1R [16 (link)] were used for amplifying and sequencing these sequences: ITS, nrLSU, and tef1, respectively. Polymerase chain reaction (PCR) reactions were performed in a total volume of 25 μL containing 0.5 μL template DNA, 11 μL distilled water, 0.5 μL of each primer, and 12.5 μL 2 × PCR mix (DreamTaqtm Green PCR Master Mix, Fermentas, MA, USA). PCR amplification reactions were performed in a Professional Standard Thermocycler (Biometra, Göttingen, Germany) as follows: denaturation at 95 °C for 4 min; 35 cycles (denaturation at 94 °C for 60 s, annealing at 53 °C (ITS and LSU)/50 °C (tef1) for 60 s, and extension at 72 °C for 60 s); and a final extension at 72 °C for 8 min. The PCR products were electrophoresed on 1% agarose gels and sequencing was performed on an ABI Prism^® 3730 Genetic Analyzer (PE Applied Biosystems, Foster, CA, USA) at the Beijing Genomic Institute (BGI). The raw sequences were assembled and edited using SeqMan implemented in Lasergene v7.1 (DNASTAR Inc., Madison, WI, USA) and deposited in GenBank.

ERIC-PCR was performed for molecular typing using the primer ERIC2 (5′-AAGTAAGTGACTGGGGTGAGCG-3′). 2 μL of DNA template were mixed with 12.5 μL of DreamTaq^TM Green PCR Master mix (Thermo Scientific, Vilnius, Lithuania), 1 μL of primer (10 pmol), and an adequate volume of sterile nuclease-free water to make a 25 μL reaction mixture. The PCR was carried out in a Bio-Rad T100™ Thermal Cycler (Bio-Rad, Hercules, CA, USA), with the following protocol: initial denaturation at 94 °C for 15 min, followed by 40 cycles of amplification. Denaturation at 95 °C for 1 min, primer annealing at 37 °C for 1 min, and primer extension at 72 °C for 1 min were used in each cycle. After the amplification cycles, samples were kept at 72 °C for 10 min to allow partially produced DNA to extend [78 (link)]. Then, 2% w/v agarose gel electrophoresis was used to visualize ERIC fragments, and the data were evaluated using GelJ v.2.0 software (Open source Java software) [83 (link),85 (link)]. Invitrogen 1 kb plus ladder (Thermo Fisher Scientific, Waltham, MA, USA) was used to normalize the fragment patterns in GelJ v.2.0 software [27 (link)].

Total RNA was extracted with NucleoSpin® RNA II (Machery-Nagel, Düren, Germany) and cDNA was produced with the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Rockford, IL, USA). GCSH full length coding sequence was amplified with primer pair fw: 5′-ATGGCGCTGCGAGTGG-3’ and rv: 5′-TCACTCCTCAATAGATTTTATG-3′ using Dream Taq^TM Green PCR Master Mix (Thermo Fisher Scientific Inc., Vilnius, Lithuania). qRT-PCR was performed with primer pair fw: 5′-GTCTCCCTGAAGTTGGGACA-3′ and rv: 5′-TCTGAAGGGTTACTCAGTGTCA-3′ using iTaq^TM SYBR^® Green Supermix (Bio-Rad Laboratories Inc., Hercules, USA) in the iQ^TM5 Multicolor Real-Time PCR Detection System (Bio-Rad, München, Germany). Relative gene expression was normalized to the GAPDH housekeeping gene: fw: 5′-CAAGGTCATCCATGACAACTTTG-3′ and rv: 5′-GTCCACCACCCTGTTGCTGTAG-3′. Sequencing of both transcript variants was done by Seqlab Sequencing Laboratories (Göttingen, Germany).

Total RNA from yeast cells was extracted using the RiboPureTM RNA Purification Yeast Kit (Life Technologies) according to manufacturer’s instructions. RNA extracts were treated with DNase for one hour at 37°C. To reverse transcribe the RNA 1st Strand cDNA Synthesis Kit for RT-PCR (Roche) was used according to manufacturer’s instructions and 2000ng of RNA in a 20 μl reaction with random hexamers was used. PCR of the resulting cDNA was performed using DreamTaqTM Green PCR master mix (Thermo Scientific) with strand-specific primers against CEN1, 3 and 8 listed in S2 Table, together with primers against CDC28 (as an internal control) in 50 μl PCR reactions. 35 PCR cycles were used.

All isolates were screened for ARGs using conventional singleplex and multiplex PCRs. Primers used along with their annealing temperature are given in Table 3. The PCR cycle conditions were as follows.
The reaction mixture contained 12.5 μL of 2X mastermix (Thermo scientific Dream Taq^Tm green PCR master mix), 0.2 μL each of forward and reverse primers (100 pmoles/µL), 2 μL of DNA template, and PCR-grade water up to the final volume of 25 μL. The cycling conditions for the PCR reaction were as follows: an initial denaturation for 10 min at 94 °C, followed by 35 cycles containing denaturation at 94 °C for 40 sec, annealing for 40 sec, initial extension at 72 °C for 1 min, and a final extension for 5 min at 72 °C. Agarose gel electrophoresis was performed at 90 V for 55 min in 1.5% agarose gel made in 10X Tris boric acid-EDTA (TBE) buffer.

Genomic DNA was extracted from the fresh leaves (100mg) of the four provenances of F. hodginsii using the cetyltrimethylammonium bromide (CTAB) method [19 ]. We used the assembled contig data (at least 200 bp) to search for hexanucleotide repeats of SSR loci. Identified SSR loci were then grouped into different classes. The SSR locus density was determined based on the frequency of SSR loci and the total length of contigs containing SSRs. Eleven pairs of SSR primers were used to analyse [20 (link)] (S1 Table). We also evaluated the motif length, loci numbers, and mean repeat numbers for the selected repetitive motifs [21 (link)].
The polymerase chain reaction (PCR) was conducted in a final volume of 20 μL containing 1 μL genomic DNA, 1 μL each of forward and reserve primer (10 μM), 10 μL of Dream Taq^TM Green PCR Master Mix (Thermo Fisher Scientific, USA), and 7 μL ddH₂O. The following PCR conditions were used: an initial denaturation of 95°C for 2 min; 35 cycles of 95°C for 30 s, annealing temperature of 53°C for 60 s, and 72°C for 35 s; followed by a 10-min extension at 72°C. The amplified products were evaluated on 5% agarose gel using the BIO-RAD Gel Doc^TM XR+. Fragment sizes were determined using Image Lab software version 6.0.