C1000 touch pcr thermal cycler

According to the manufacturer’s instructions, total RNA was extracted from approximately 50 mg of colon tissue using the TRIzol reagent, and its quantity was determined using spectrophotometry at 260 nm. The Superscript III first-strand synthesis method was used to generate complementary DNA (cDNA) from 2.5 μg of total RNA (Invitrogen, Carlsbad, CA, USA). QPCR was performed using SYBR Green reagents on a C1000 Touch PCR Thermal Cycler (BIO-RAD Laboratories, Shanghai, China). The primer sequences are listed in Supplementary File S1. mRNA levels were calculated using the 2^−ΔΔCT method and normalized to GAPDH levels.

Genomic DNA of various bacterial species was extracted using a standard DNA purification protocol (32 (link)). PCR was performed using Taq DNA polymerase with Standard Taq Buffer (M0273, New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocol. Briefly, PCRs were prepared to contain final concentrations of 200 μM dNTPs, 0.2 μM each primer (Table 4), 0.025 U/μl Taq DNA polymerase, and 1 ng/μL of bacterial genomic DNA. Thirty cycles of 95°C for 30 s, 60°C for 1 min, and 68°C for 1 min were performed using a C1000 Touch PCR Thermal Cycler (Bio-Rad, Hercules, CA, USA). PCR products were visualized by agarose gel electrophoresis followed by ethidium bromide staining. Alternatively, Luna qPCR Master Mix (M3003, New England Biolabs) was used according to the manufacturer’s protocol. Briefly, qPCRs were prepared to contain final concentrations of 0.25 μM each primer and 1 ng/μL of bacterial genomic DNA. Forty-five cycles of 95°C for 15 s and 60°C for 30 s were performed using a Bio-Rad CFX96 real-time PCR machine. Fluorescence signals indicative of the presence of PCR products were measured.

Genomic DNA was extracted from mycelium using the Plant/Fungi DNA Isolation Kit (Norgen Biotech Corp., Thorold, Canada) with the following modifications: fungal tissue was vortexed for 15 minutes with 1 mm glass beads and 500 μL lysis buffer and 1 μL RNase A prior to incubation at 65°C. After incubation on ice, the fungal mixture was centrifuged at 10,000 rpm to separate the lysate from the beads and biomass. During the column wash, the resin was dried by spinning for 10 minutes at 14,000 rpm. DNA was eluted at 10,000 rpm for 2 minutes. PCR amplifications were executed using C1000 Touch PCR thermal cycler (Bio-Rad, Hercules, USA) under conditions described in the references for each region. The internal transcribed spacer (ITS) region was amplified with the primer pair ITS1/ITS4 [20 ]. The primer pair LR0R/LR5 [21 (link)] was used to amplify the large subunit rRNA gene (LSU). The partial actin (ACT) region was amplified with the primer pair ACT512F/ACT783R [22 (link)], and the primer pair EF1-728F/ EF1-986R [22 (link)] was used to amplify partial translation elongation factor 1-alpha (tef1-α) gene sequences. The quality of the PCR products was examined using electrophoresis in 1% agarose gel. Sanger sequencing was carried out at Genome Quebec’s Sequencing Facility (Montreal, Canada).

The fungal mat from the culture was chopped into fine pieces in a sterile Petri dish and transferred to a 1.5-mL microcentrifuge tube. Genomic DNA was extracted using a NucleoSpin Plant II kit (740770.250, Macherey-Nagel) as per the manufacturer’s instructions. Polymerase chain reaction (PCR) was set up using DreamTaq Green PCR Master Mix (2×) (K1081, Thermo Fisher Scientific), along with 28S ribosomal RNA (rRNA) primers (forward primer: 5′-GTGAAATTGTTGAAAGGGAA-3′; reverse primer: 5′-GACTCCTTGGTCCGTGTT-3') as described earlier.¹⁴ (link) Polymerase chain reaction amplification was carried out on a C1000 Touch PCR thermal cycler (Bio-Rad Technologies) under conditions of initial denaturation at 94°C for 4 minutes followed by 34 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 1 minute, extension at 72°C of 1 minute, and final extension at 72°C for 1 minute. The PCR products were subjected to agarose gel electrophoresis with 1% agarose gel and the amplicons were purified using a QIAquick PCR purification kit (28106, Qiagen) according to the manufacturer’s protocol and sequenced using BigDye Terminator v. 3.1 (Applied Biosystems). The sequences were then subjected to BLAST search for species identification and submitted to the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and accession numbers were obtained (Table 1).

According to the manufacturer’s instructions, total RNA was extracted from approximately 50 mg of colon tissue using the TRIzol reagent, and its quantity was determined using spectrophotometry at 260 nm. The Superscript III first-strand synthesis method was used to generate complementary DNA (cDNA) from 2.5 μg of total RNA (Invitrogen, Carlsbad, CA, USA). QPCR was performed using SYBR Green reagents on a C1000 Touch PCR Thermal Cycler (BIO-RAD Laboratories, Shanghai, China). The primer sequences are listed in Supplementary File S1. mRNA levels were calculated using the 2^−ΔΔCT method and normalized to GAPDH levels.