Biorad t100 thermal cycler

The colorimetric LAMP assays were optimized to provide a clear visual detection based on the drop in pH caused by DNA target synthesis. When using the neutral red dye, the pH drop produces a change in the colour solution from yellow to fuchsia. For this, the reaction mixtures were prepared at a final volume of 25 μL, containing 0.5 × Bst DNA polymerase reaction buffer (10 mM Tris–HCl, 5 mM (NH₄)₂SO₄, 25 mM KCl, 1 mM MgSO₄, 0.05% Tween 20, pH 8.8 New England Biolabs Inc., MA, USA), 0.2 μM each external primer F3 and B3, 2 μM each inner primer FIP and BIP, 12U Bstv2.0 DNA polymerase (New England Biolabs Inc., MA, USA), 1.4 mM of each dNTPs, 0.003% of neutral red dye and 5 μL of a test sample. The reaction mixture for the Pvr47-LAMP was adjusted to 6 mM MgSO₄, the PvCOX1-LAMP was adjusted to 6 mM MgSO₄ and 0.4 M betaine, the Pfr364-LAMP was adjusted to 8 mM MgSO₄ and the PfEMP1-LAMP was adjusted to 8 mM MgSO₄ and 0.4 M betaine.
A positive reaction was assessed by visual inspection and considered as such only if the reaction turned from yellow to fuchsia after 65 min of incubation at 60 °C. Incubation steps were performed on a Bio-Rad T100 TM Thermal Cycler. (Bio-Rad Laboratories, CA, USA).

Total RNA was isolated from the VICs using a RNeasy kit (Qiagen, Germantown, MD, USA) following the manufacturer’s instructions. First-strand cDNA was then synthesized using the RevertAid First strand cDNA Synthesis kit (Thermo Scientific, EU) in a BIO-RAD T100TM-Thermal Cycler (Bio-Rad, Hercules, CA, USA). qPCR analysis was carried out using SYBR Green PCR master mix (Roche, Penzberg, Germany) in a Light Cycler 480 Real time-PCR Detection system (Roche), according to the manufacturer’s protocol. The results were presented as the ratio of target PCR products relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as an internal control. The relative expression of the target genes was calculated utilizing the comparative threshold cycles (2^−ΔΔCt) method.

Genomic DNA was extracted from blood samples of patients firstly diagnosed with Rb using the iPure blood genomic DNA kit (IGEbio, CZ313-S). The extracted DNA was quantified using Nanodrop 2000 (Nanodrop, Wilmington, DE, USA). The primers used for detecting RB1 mutations (shown in Supplementary Table S1) were designed using Primer Premier 3.0 software for 26 translating exons and the adjacent intron–exon regions of the RB1 gene, except exon 15. The PCR had a total volume of 20 µl, including 10 µl HiFipfu polymerase (IGEbio, P300-S), 7 µl nuclease-free water, 1 µl forward and reverse primers (10 uM) each, and 2 µl DNA template. A BIO-RAD T100 TM Thermal cycler (Bio-Rad, Gladesville, Australia) and a touchdown PCR protocol were used for amplification. The PCR conditions were as follows: initial denaturation at 94 °C for 5 min followed by 10 cycles of denaturation at 94 °C for 30 s, annealing at 65 °C for 30 s, with the temperature of the heating block decreased by 1 °C per cycle, and then extension at 72 °C for 40 s. This was then followed by 25 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 40 s and final extension at 72 °C for 3 min.

The mycelia of the two strains were inoculated into the flask containing 200 mL of PDB medium, and the mycelium biomass of about 100 mg was harvested after incubation on the shaker (160 rpm, 28 °C, 3 days). According to the research of Guo et al. (2001 (link)), the genomic DNA of endophytic fungi was isolated and extracted by the CTAB method.
Fungal identification was based on their ITS sequences and TEF-1α sequences, which was accomplished by PCR amplification with the universal primers (V9D 5′-TTA AGT CCC TGC CCT TTG TA-3′; LS266 5′-GCA TTC CCA AAC AAC TCG ACT C-3′) and TEF-1α primers (TEF1-728 F 5′-CAT CGA GAA GTT CGA GAA GG-3′; TEF1-rev 5′-GCC ATC CTT GGA GAT ACC AGC-3′) (Van Den Gerrits and Hoog 1999 ; Carbone and Kohn 1999 (link)). Reaction volumes of 50 µL contained 1 mM dNTPs, 1.75 units of Taq DNA polymerase, 0.2 µg of genomic DNA, 1.5 mM MgCl₂, 5 µL 10 × PCR buffer and 20 pM of each primer. The mixed samples were amplified in Bio-RadT100TM Thermal Cycler (Bio-Rad, USA) to obtain PCR products. The 5 µl PCR product was verified by electrophoresis display on 1% (w/v) agarose gel in 1× TAE buffer (1 mmoL/L EDTA, 40 mmoL/L Tris, pH 8.0). Then the same primers were used for sequencing (Shanghai Sangon Biologic Engineering Technology and Service Co., Ltd., Shanghai, People's Republic of China).

RNA was extracted from leaf and stem samples using a ZR Plant RNA MiniPrep™ kit (Zymo Research, Irvine, CA, USA). RNA samples were quantified on a NanoDrop Lite Spectrophotometer (Thermo Scientific™, Waltham, MA, USA) and 1 µg RNA of each sample was treated with DNaseI Amplification Grade (Sigma-Aldrich, St. Louis, MO, USA) to remove genomic DNA contained in the sample. After a heat step to deactivate the DNaseI (70 °C for 10 min in the presence of 50 mM EDTA to prevent denaturation), samples were immediately reverse transcribed to cDNA using iScript^TM cDNA Synthesis Kit (BioRad Laboratories, Hercules, CA, USA).
To test that the cDNA reaction was successful and to confirm that no genomic DNA remained in the samples, a test PCR was performed using primers designed over an intron. PCR reactions were set up using MyTaq^TM DNA Polymerase (Bioline, Alexandria, NSW, Australia) and were run in a BioRad T100^TM Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) with the following protocol: 95 °C for 1 min, followed by 35 cycles of a denaturing step of 95 °C for 15 s, an annealing step of 51 °C for 15 s, and an extension step of 72 °C for 1 min. The protocol finished with a final extension step of 72 °C for 10 min. Reactions were run on a 2% agarose gel and imaged with a Chemidoc XRS+ with Image Lab™ software (Bio-Rad Laboratories, Hercules, CA, USA) to visualize PCR products.