Mangotaq

The DNA of the R. mucilaginosa isolate obtained on SDA control media was extracted using the Animal and Fungi DNA Preparation Kit^® (Jena Bioscience) according to the manufacturers’ instructions. The ITS1 and ITS2 primer sets targeting the ITS (Internal Transcribed Spacer) region were used for identification⁴⁶ . For PCR amplification, the following mixture was used in a 25 µL final volume: 5 µL of 5X MangoTaq Colored Reaction Buffer (Bioline), 1.25 µL of 50 mM MgCl₂ (Bioline), 0.5 µL of 10 mM dNTP (Bioline), 1.25 µL of each primer (Macrogen Sequencing Service, Korea), 0.25 µL of 5 U/µL MangoTaq (Bioline) and 2 µL of DNA. Negative control samples were used for each pair of primers. The amplification series consisted of 35 cycles of the following: 95 °C for 30 s, 56 °C for 30 s and 72 °C for 30 s. The fragment obtained after amplification was sequenced at Macrogen (Macrogen Sequencing Service, Korea).

The final method is described in the following: The nested PCR was carried out in 50 μl using a standard reaction buffer, 3.0 mM of MgCl_2, 200 μM of each dNTP, 50 pmol of each primer and 1 U of Taq polymerase (MangoTaq, Bioline, London, UK). Primary PCR of the D8 region (~1000 bp) of the LSU gene was conducted using the primer pair LSU2040F/LSU3020R, followed by secondary (nested) PCR (~500 bp) using the primer pair LSU2065F/LSU2557R. The conditions of the primary PCR were: 94 °C for 5 min (initial denaturation), followed by 35 cycles of 94 °C for 30 s (denaturation), 58 °C for 30 s (annealing) and 72 °C for 50 s (extension), with a final extension of 72 °C for 5 min. The conditions of secondary PCR were the same, except that the extension step was 30 s instead of 50 s. Except for the no-template controls, 2 μl of genomic DNA were added to the primary PCR, from which 1 μl was carried over to the secondary PCR. No-template (negative) controls were included at all steps, and no-template controls were carried over from the primary to the secondary (nested) PCR. A well-known positive control sample (C. parvum DNA) was included in each PCR run. The sequencing of LSU amplicons was performed (as described for SSU amplicons) using primers LSU2065F and LSU2557R.

Routine PCR was conducted in a total volume of 20 µl using Mango Taq (Bioline) master mix. Cycling conditions comprised an initial denaturation step at 94°C for 2 min 30 s, followed by 30 cycles of denaturation (94°C for 30 s) annealing (60°C for 30 s) and extension (72°C for 5 min). An EmeraldAmp Taq polymerase (Master Mix) was used in long-range PCR to bridge Illumina sequence scaffolds. Primer sequences and amplicon sizes are listed in electronic supplementary material, table S1.

Peripheral blood was collected from all participants in EDTA tubes. Genomic DNA was isolated from white blood cells using the QIAamp DNA Blood Mini Kit (QIAGEN). IL-8 polymorphisms were determined by analyzing fragment length polymorphisms of the respective PCR products (PCR-RFLP). The amplicons were generated from 25 ng genomic DNA as template in a 25 µL reaction mix, containing 5 pmol of the respective forward and reverse primers (Table 1) and MangoMix™ (Bioline) providing MangoTaq™ DNA polymerase, MgCl₂, and dNTPs. The amplification was carried out after an initial hot start at 95 °C for 5 min, for 45 cycles starting with a 30 s denaturation at 95 °C, followed by a 30 s annealing at the temperature given in Table 1, and a 60 s extension at 72 °C. After a final extension step at 72 °C for 7 min, the PCR products were digested with the respective restriction endonuclease (all from New England Biolabs, Ipswich, MA, USA) under the conditions given in Table 1. The restriction fragments were separated with capillary electrophoresis using the Fragment Analyzer™ Automated CE System (Advanced Analytical, Ankeny, IA, USA) and the DNF-905 dsDNA Kit (Agilent, Santa Clara, CA, USA). The sizes of the fragments were assessed using the software PROSize^® 3.0 version 3.0.1.6 (Advanced Analytical Technologies).

The genetic diversity within 111 strains isolated from spontaneously fermented Negro Saurí juice and one commercial wine Saccharomyces cerevisiae (Cross Evolution, Lallemand) was evaluated by PCR amplification of inter-delta (δ) regions, which flank Ty elements in the yeast genome^{58 (link)}. DNA isolation was performed according to Liu et al.^{103 (link)}. PCR amplification of inter-delta sequences was with primers δ12 (5′-TCAACAATGGAATCCCAAC-3′) and δ21 (5′-CATCTTAACACCGTATATGA-3′)^{57 (link)}. PCR amplification was performed in a 25 µl reaction using 30–100 ng template DNA, 0.8 µM each primer, 0.2 mM dNTP and 2.5 unit of Mango Taq (Bioline). PCR conditions were as follows: initial denaturation for 4 min at 95 °C followed by 35 cycles of 30 s at 95 °C; 30 s at 46 °C and 90 s at 72 °C, and a final extension step of 10 min at 72 °C. The PCR products were separated on 2% agarose gels and the size of the amplified DNA fragments estimated with 50 bp HyperLadder DNA markers (Bioline).
PCR amplification was repeated four times per isolate to ensure repeatability, with the results considered accurate when 3 or 4 replicates were equal; where this did not occur, the results were reviewed.

Genomic DNA was isolated from each LCL using the NucleoSpin Tissue Kit (Macherey-Nagel). PCR products with the CAG repeats from HTT were produced with primers oVIN-1333 and oVIN-1334 (table S5). PCR products containing the CTG repeats from DMPK were amplified with primers oVIN-1252 and oVIN-1251 (table S5). For normal-length alleles, several PCRs were set up with MangoTaq (Bioline), and the products were gel-extracted and Sanger-sequenced with the same primers used for the amplification. For expanded alleles, small-pool PCRs were performed on the basis of a previously described protocol (43 (link)). Briefly, the same primers were used for the amplification of expanded DMPK and HTT alleles with 1 ng of genomic DNA per PCR. The products were run on an agarose gel and transferred to a nylon membrane. An oligo made up of 10 CAGs was used to obtain a radioactive probe used for the visualization of the expanded alleles. The number of repeats reported here is an estimation of the modal number of repeats.