Mgcl2

sj and DβTRECs were co-amplified together with the CD4 gene used as a single copy gene in a step called “pre-PCR,” using outer (out) primers (Table 1). Briefly, 10 μl of cell lysates or plasmids, containing sjTRECs (or DβTRECs) with CD4 inserts, were added to 90 μl of a mix composed of 1 μl of each primer at 100 mM, 20 μl of 5× colorless GoTaq® Flexi buffer (Promega), 14 μl of MgCl₂ (25 mM, Promega), 4 μl of deoxynucleoside triphosphate (dNTP, 10 mM, Promega), 0.8 μl of GoTaq® Flexi DNA Polymerase (4U, Promega), and completed with nuclease-free water (Ambion). Amplification was performed in an iCycler thermocycler (Bio-Rad), and the PCR conditions were: initial denaturation at 95°C for 10 min; 22 cycles of amplification each consisting of 30 s at 95°C, 30 s at 60°C, and 2 min at 72°C, followed by final elongation at 72°C for 10 min and cooling at 20°C.

Viral genomic DNA was amplified using primers designed by Snapgene software (https://www.snapgene.com), except F3 and F5 primers were obtained from references [11 (link), 12 (link)] to amplify five overlapping amplicons that encompassed the whole genome sequence of the virus (Table 1). Amplification of the viral genome was achieved using 1X GoTaq® G2 Green Master Mix (reaction buffer (pH 8.5), 200 µM dATP, 200 µM dGTP, 200 µM dCTP, 200 µM dTTP and 1.5 mM MgCl₂, (Promega, USA)), 0.4 µM of each forward and reverse primers and 100 ng of templates DNA in a total volume 50 μl of reaction mixture. The cycling rounds consisted of 35 cycles of denaturation at 95°C for 30 s, annealing for 30 s at different temperatures depending on the primer, and extension time at 72°C for 1 min to extend X, PreC-C, and P fragments as well as 1.5 min to extend PreS1-S and P1 fragments. Initial denaturation was performed at 95°C for 2 min, and a final extension was performed at 72°C for 5 min in a thermal cycler (Prime Elite Satellite, Techne, UK). Finally, the amplicons were sent for Sanger sequencing (Macrogen, South Korea).

The whole cyp51A gene and its promoter were sequenced in both strands from all A. fumigatus strains with elevated MIC values, using five sets of primers: PA5 and PA7 (Mellado et al., 2001 (link)), AF306F and AF855R, AF766F and AF1330R, AF1179F and AF1709R, and AF1426F and AF2025R (Alanio et al., 2011 (link)).
The PCR mixture contained 5 μL of DNA extract and 20 μL of mix composed of 0.625 U of GoTaq^® Hot Start Polymerase (Promega), 1x Colorless GoTaq^®Flexi Buffer (Promega), 2 mM of MgCl₂ (Promega), 0.8mM of dNTP mix (Eurobio), and 0.2 μM of each primer. The amplification program consisted of 5 min at 94°C, 30 cycles of 30 s at 94°C, 30 s at 58°C, and 1 min at 72°C, followed by a final step of 10 min at 72°C.
Following purification and sequencing as described above, sequences of resistant strains were compared to the wild-type A. fumigatus sequence CM 237 (GenBank accession number AF338659), at http://blast.ncbi.nlm.nih.gov/Blast.cgi.

A total of 16 decamer universal RAPD primers (synthesized by First Base Laboratory, Serdang, Malaysia), as shown in
Table 1, were used to amplify the DNA samples of the three Malaysian endemic
Coelogyne species. These sequences were obtained from published articles (
Table 1) where they have been used successfully in other orchid species. The RAPD amplification reactions were performed in a 10 μl volume containing 50 ng of plant DNA, 1.0 μM of RAPD primer (First Base Laboratory), 1 × PCR buffer (Promega, USA), 2.5 mM of MgCl
₂ (Promega, USA), 0.1mM dNTPs mix (Promega, USA), 0.5 units of Taq DNA polymerase (Promega, USA), using a thermal cycler machine (Eppendorf Master Cycler Gradient, Hamburg, Germany), The run conditions were: 1 cycle of initial denaturation at 95°C for 10 min; 45 cycles of denaturation at 95°C for 1 min, annealing at 35°C for 2 min and extension at 72°C for 2 min; and 1 cycle of final extension at 72°C for 10 min. The amplicons were electrophoresed on 1.2% TBE (Tris-borate-EDTA) agarose gel for 90 minutes at 70 V and stained with 0.5μg/mL ethidium bromide. The gel was visualised under a UV transilluminator (UVIdoc, USA) and the gel image was taken using a camera (UVItec, UK). Species-specific bands were identified from the bands produced during the RAPD amplifications.

We tested a total of 24 different ISSR markers. The selection of markers for this study was based on the identification of a particular band profile to facilitate the identification of each morphospecies, in addition to presenting good resolution and a high number of bands. Two ISSR markers were retained for our study: (AG)₈Y and (GA)₈C (Table 2).
PCR amplifications were performed in 15 μl reaction volume containing ~20 ng of template DNA, 1.5 μl 5X Green Buffer (Promega), 200 μM dNTP (dNTP mix; Promega), 3 mM MgCl₂ (Promega), 1 μM of primer (Integrated DNA Technologies), and 1.25 U GoTaq Flexi DNA Polymerase (Promega); finally, the volume was adjusted with ultrapure water. Amplifications were conducted in a T100 Thermal Cycler (Bio-Rad™): initial denaturation step at 94°C for 4 min, 39 cycles of denaturation at 94°C for 45 s, annealing temperature (Ta) 54°C or 56°C depending on the ISSR primer (Table 2), extension temperature at 72°C for 2 min, and a final extension at 72°C for 10 min. DNA banding patterns were visualized by electrophoresis, performed with 3 μl of amplified products on a 2% agarose gel using 1X TAE buffer and post-staining with GelRed™ (Biotium), at 110 V for 2 h. A 100 bp DNA Ladder (Promega) was used to estimate amplification product lengths. Fragment (band) patterns were visualized and digitized using an imaging system (PhotoDoc-it, UVP®).