Peltier thermal cycler ptc200

Quantitative real-time PCR (qRT-PCR) was performed using the SYBR® Premix Ex Taq™ II (Perfect Real Time) kit (Takara, Japan) on a Peltier Thermal Cycler PTC200 (Bio-Rad, USA) with gene-specific primer pairs (Table S1). The related expression level was normalized against the expression level of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in tea plant^{48 (link)} or actin in tobacco^{49 (link)}. The melting curve was performed to determine the PCR product size and to detect possible primer dimers. Triplets of all samples were run. The cycle number at which the reaction crossed an arbitrarily placed threshold (CT) was determined for each gene, and the relative expression of each gene was determined using the equation 2^−ΔΔCT, where ΔΔCT = (CT_Target − CT_GAPDH/Actin)_sample − (CT_Target − CT_GAPDH/Actin)_control^{50 (link)}.

Genotyping was accomplished by isolating genomic DNA from tail tips, using the INVISORB spin tissue mini kit (Stratec, Berlin, Germany) and quantified afterward by PCR. The following primers were used: Klk7 loxP sites, 5′-GGGATGTAGGATTATGAGTGAGC-3′ (forward) and 5′-CAGTCCAGTGAACTGCTCACC-3′ (reverse), as well as Cre-recombinase, 5′-GCGGTCTGGCAGTAAAAACTATC-3′ (forward) and 5′-GTGAAACAGCATTGCTGTCACTT-3′ (reverse). PCR was performed for 35 cycles, 95 °C for denaturation (loxP sites and Cre), 60 °C (loxP sites) or 56 °C (cre) for annealing and 72 °C (loxP sites and Cre) for elongation performed with the Fermentas Dream Taq Polymerase (Fermentas, St. Leon-Rot, Germany) and a Peltier Thermal Cycler PTC-200 (Bio-Rad, Hercules, CA, USA). With DNA from control mice, a 276 bp band and with DNA from ATKlk7^−/− mice a 402 bp band were obtained on agarose gel.

In August 2020, fungal macroforms were observed only in the La Concha orchard. Internal samples were obtained in the laboratory from two fruiting bodies of different colors, and DNA extraction was performed using a commercial kit (Ultraclean Isolation DNA Kit, MoBio brand, San Mateo, CA, USA) following the manufacturer’s instructions. The quality and concentration of the DNA were verified using a Nanodrop 2000c spectrophotometer. The internal transcribed spacer (ITS) gene region, 18S rDNA (partial sequence) gene, 5.8S rDNA gene, internal transcribed spacers 1 and 2 (full sequence), and the 28S rDNA gene (partial sequence) were amplified by PCR using the ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) [42 ] and ITS4 5′-TCCTCCGCTTATTGATATGC-3′) primers [42 ].
The protocol included an initial denaturation at 94 °C for 1 min, followed by 40 cycles each of 94 °C (1 min), 50 °C (2 min), and 72 °C (1 min), and a final extension at 72 °C (5 min). PCR was conducted using a Peltier Thermal Cycler PTC-200 (Bio-Rad, Hercules, CA, USA), and the PCR products were verified by electrophoresis on a 1.2% agarose gel. PCR products were sequenced in both directions using an Applied Biosystems Model 3730XL Automated DNA Sequencing System.