Total genomic DNA was extracted from abdominal muscle tissue following the treatment of a standard proteinase K, phenol–chloroform extraction, and ethanol precipitation [26 ]. Two different fragments (16S rRNA and COI) in mtDNA were amplified and sequenced. The 16S rRNA and COI sequences were amplified using these 1471 (5’-CCT GTT TAN CAA AAA CAT-3’) and 1472 (5’-AGA TAG AAA CCA ACC TGG-3’) [27 ] and COI-F (TTT ATC TTC GGA GCG TGA GC) and COI-R (AGT TAT TCC TGG GGC TCG TAT G) [28 ] primers, respectively. Thermal cycling was performed on GeneAmp 2400 thermal cycler (Perkin-Elmer, Norwalk, CT, USA) and PCR conditions consisted of 39 cycles of denaturation at 95°C for 50 s, annealing at 50°C for 1 min, and extension at 72°C for 1.5 min. An initial denaturation step at 95°C for 5 min and a final extension holding at 72°C for 10 min were respectively included in the 1st and last cycles. PCR product was separated by electrophoresis on 1.5% agarose gels, purified with the Gene Clean II kit (Bio101, Vista, CA, USA), and sequenced on an ABI 377 DNA sequencer (Applied Biosystems, Inc.; Foster City, CA, USA).
Geneamp 2400 thermal cycler
Manufactured by PerkinElmer
Sourced in United States
The GeneAMP 2400 Thermal Cycler is a laboratory instrument designed for DNA amplification and analysis. It provides precise temperature control and automated cycling capabilities to facilitate the polymerase chain reaction (PCR) process. The device features a compact and user-friendly design to support various molecular biology applications.
Automatically generated - may contain errors
3 protocols using geneamp 2400 thermal cycler
1
Mitochondrial DNA Extraction and Sequencing
2
RNA Isolation and cDNA Synthesis from Endometrial Tissue
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RNA from normal and cancer tissue was isolated using the total RNA isolation kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer's instructions and quantified spectrophotometrically. The isolation of RNA from endometrial lesions was performed followed by overnight tissue sample incubation with Proteinase K enzyme at 37°C. First-strand cDNAs were obtained by the reverse transcription of 1 mg of total RNA using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer's protocol. The reverse transcription polymerase chain reaction (RT-PCR) was carried out in a GeneAMP 2400 Thermal Cycler (Perkin-Elmer, USA) according to the following thermal profile: 10 min at 25°C, 120 min at 37°C, and 5 min at 85°C.
3
RNA Isolation and Reverse Transcription Procedure
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Total RNA from normal and cancer tissue samples was isolated using Trizol ® Reagent (Sigma Aldrich, USA) according to manufacturer's protocol and quantified spectrophotometrically. The reverse transcription of 2 µg of RNA was performed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA) following manufacturer's instructions. The reverse transcription polymerase chain reaction (RT-PCR) was performed in a thermocycler GeneAMP 2400 Thermal Cycler (Perkin-Elmer, USA) according to the following thermal profile: 10 min at 25°C, 120 min at 37°C, and 5 min at 85°C.
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