After 6 h of cultivation, total RNA was extracted from liver slices by Sepasol (Nacalai Tesque Inc., Kyoto, Japan). The extracted RNA was dissolved in TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA). For each sample, RNA concentration was estimated using Gene Quant Pro (Amersham Pharmacia Biotech, Cambridge, UK). The RNA was reverse-transcribed using ReverTra Ace (Toyobo Co. Ltd., Osaka, Japan). The reaction mixture (10 µL) contained 1 µg of total RNA, 1× RT buffer, 1 mM dNTP mixture, 20 U RNase inhibitor, 0.5 µg of oligo (dT) 20 primer, and 50 U ReverTra Ace. Reverse transcription was carried out at 42°C for 30 min, followed by heat inactivation at 99°C for 5 min using an Applied Biosystems 2720 thermocycler (Applied Biosystems, Foster City, CA, USA)
Applied biosystems 2720 thermocycler
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Applied Biosystems 2720 Thermocycler is a laboratory instrument used for thermal cycling, a process essential for various DNA-based applications such as polymerase chain reaction (PCR). The device precisely controls the temperature and duration of each step in the thermal cycling process, enabling the amplification of targeted DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Genequant pro, by Cytiva (1 mentions)
Revertra ace, by Toyobo (1 mentions)
Sepasol, by Nacalai Tesque (1 mentions)
Taq dna polymerase, by Enzynomics (1 mentions)
Dreamtaq, by Thermo Fisher Scientific (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Genelute bacterial genomic dna kit, by Merck Group (1 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (1 mentions)
Sanger sequencing, by Beckman Coulter (1 mentions)
4 protocols using applied biosystems 2720 thermocycler
1
RNA Extraction and Reverse Transcription from Liver Slices
2
Amplification and Sequencing of mtDNA HVSI
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The extracted DNA was used as a template for PCR amplification of the 451-bp fragment at the 15,974-16,425-bp region of mtDNA, containing HVSI. The 25-µL PCR mixture was comprised of 1X Taq buffer, 2.5 mM dNTPs, 2.0 mM MgCl 2 , 10 pM each of the forward and reverse primers, 2.5 U Taq DNA polymerase (Enzynomics, Korea, Cat. P050B), and 30-40 ng template DNA. The following primers were used for PCR amplification: forward, 5'-CTCCACCATTAGCACCCAAAGCTAAG-3' and reverse, 5'-GATATTGATTTCACGGAGGATGGTGGTC-3'. The reaction was performed in an Applied BioSystems-2720 thermocycler (Applied Biosystems, Foster City, CA); the reaction conditions were set as follows: initial denaturation at 94°C for 4 min; 35 cycles of denaturation at 94°C for 40 s, annealing at 56°C for 1 min, and extension at 72°C for 1 min, and a final extension at 72°C for 5 min. The nucleotide sequences of purified PCR fragments obtained from individuals of different tribal populations were commercially analyzed by Macrogen (Geumcheon, South Korea).
3
Multiplex PCR Assay for Colletotrichum Identification
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DNA from mycelia of 7-day-old Colletotrichum cultures was extracted using the DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s instructions. A multiplex PCR assay was performed to differentiate isolates of the CGSC and CASC by partial amplification of the GAPDH and CAL genes using primer pairs GDF1/C-GAPDH-R, CALF1/Cg-R, and CALF1/Ca-R149 (link). PCR amplifications were carried out in 25 μL volumes containing 10X PCR buffer (includes 20 mM MgCl2) (Dream Taq, Thermo Fisher Scientific, Waltham, MA, US), 200 ng of gDNA, 2 mM dNTP, 1 u/μL of Taq DNA polymerase (Dream Taq, Thermo Fisher Scientific, Waltham, MA, US) and 10 μM of each primer using Applied Biosystems 2720 Thermo Cycler (Thermo Fisher Scientific, Waltham, MA, US). Cycling conditions were as follows: initial denaturation of 4 min at 94 °C, followed by 35 cycles of denaturation at 94 °C for 40 s, annealing at 56 °C for 40 s and extension at 72 °C for 1 min, with a final extension at 72 °C for 5 min. PCR products were visualized in 1% (w/v) agarose gels in 1xTAE buffer electrophoresed at 94.1 V for 45 min.
4
16S rRNA Gene Amplification and Sequencing
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Genomic DNA of isolates which produced clear zones of inhibition was extracted from culture cell pellets using the GenElute™ Bacterial Genomic DNA Kit (Sigma-Aldrich; Co. Wicklow, Ireland). Extraction of DNA was confirmed by agarose gel electrophoresis and subsequently the 16S rRNA gene was amplified using the following 16S eubacterial primers CO1; 5’-AGTTTGATCCTCCTGGCTCAG-3’ and CO2; 5’-TACCTTGTTACGACTT-3’.55 (link) The DNA was amplified with Invitrogen Platinum PCR Supermix (ThermoFisher Scientific, Dublin, Ireland) and PCR reactions performed on the Applied Biosystems 2720 Thermocycler (ThermoFisher Scientific, Dublin, Ireland). The amplification cycle used was as follows: 94°C for 2 min, and 30 cycles of the following: 94°C for 30 s, 50°C for 30 s and 72°C for 1.5 min. The purity and quantity of DNA present was checked on the NanoDrop 1000 (ThermoFisher Scientific, Dublin, Ireland) and the PCR product was then purified using the QIAquick™ PCR Purification Kit (Qiagen; Manchester, UK). The complete sequence of the 16S rRNA gene was determined by Sanger sequencing (Beckman Coulter, Essex, UK). The species was putatively identified by comparing the resulting sequence with deposited species in the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi ) with a high percentage nucleotide identity (>98%).
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