Tprofessional thermocycler

Manufactured by Analytik Jena

Sourced in Germany, New Zealand, Japan

The TProfessional Thermocycler is a high-performance laboratory instrument designed for DNA amplification and other temperature-controlled applications. It features a compact design, intuitive user interface, and precise temperature control to ensure consistent and reliable results.

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39 protocols using tprofessional thermocycler

mRNA Extraction and Reverse Transcription

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Following the suppliers protocol, 20 µl of mRNA extract were digested with 2 µl of 50 x Turbo DNAfree Buffer and 1µl of Turbo DNAse from the TURBO DNA-free ™ Kit (Ambion). The reaction was carried out at 37 °C for 45 min in a thermocycler (Tprofessional Thermocycler by Biometra or Flexcycler by Analytik Jena). DNAse was inactivated by adding 2.3 µl of TurboDNA-free Inactivation reagent. Samples were centrifuged at 11,000 g for 1.5 min, and the supernatant was transferred into a clean 1.5 ml DNA LoBind reaction tube (Eppendorf). Aliquots were taken for reverse transcription and qPCR control reactions.
Reverse transcription of mRNA was performed with the ThermoScript™ RT-PCR System (Life Technologies). 5 µl of DNA-free mRNA were added to 0.6 µl primer AF-Endo reverse (see section 2.4), 2 µl of 10 mM dNTP Mix and 4.4 µl of DEPC-water, leading to a total volume of 12 µl. After RNA denaturation at 65 °C for 5 min in a thermocycler (Tprofessional Thermocycler by Biometra), the mixture was placed on ice, and 8 µl of reverse transcription mastermix comprising 4 µl 5x cDNA synthesis buffer, 1 µl 0.1 M DTT, 1 µl RNase Out™ (40 U/µl), 1 µl DEPC-water and 1 µl of ThermoScript™ RT (15 U/µl), was added. Reverse transcription was performed at 51 °C for 60 min and stopped by termination at 85 °C for 5 min. cDNA was stored at -20°C until further analysis.

Dollhofer V., Callaghan T.M., Griffith G.W., Lebuhn M, & Bauer J. (2017). Presence and transcriptional activity of anaerobic fungi in agricultural biogas plants. Bioresource technology, 235.

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16S rDNA V3 Region Amplification Protocol

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The 16S rDNA V3 region was amplified using a hot-start touchdown protocol with primers specific for conserved regions of the 16S rRNA gene [18 (link)]. The reaction mixture contained 2 μL of genomic DNA, 25 pmol of each primer, 4 μL of dNTPs, 5 μL of 10х Ex Taq buffer, 0.5 μL of TaKaRa Ex Taq polymerase (TaKaRa, Dalian, China), and sterile deionized water to a total volume of 50 μL. To minimize heteroduplex formation, five-cycle reconditioning PCR was conducted with 5 μL of amplification mixture in a fresh reaction mixture as previously described [19 (link)]. Profiles were generated using a TProfessional Thermocycler (Biometra, Göttingen, Germany). Amplified products were confirmed by agarose gel electrophoresis (1%), and their concentrations were measured using a NanoDrop ND-1000 spectrophotometer (Nano-Drop Technologies, Wilmington, DE, USA). All amplified products were stored at -20°C until DGGE analysis.

Hu X., Zhang H., Lu H., Qian G., Lv L., Zhang C., Guo J., Jiang H., Zheng B., Yang F., Gu S., Chen Y., Bao Q., Yu L., Jiang X., Hu Q., Shi H., Gao H, & Li L. (2016). The Effect of Probiotic Treatment on Patients Infected with the H7N9 Influenza Virus. PLoS ONE, 11(3), e0151976.

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RNA to cDNA Conversion Protocol

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500 ng of RNA was typically used to generate cDNA libraries from each sample. The RNA was mixed with 8.3 μg/ml of random primers (Promega) and 0.83 mM of dNTP mix (Thermo Fisher Scientific) in a final volume of 12 μl. Parallel aliquots for each sample were prepared which did not receive reverse transcriptase and were used as negative controls for genomic DNA contamination. The samples were then incubated at 65 °C for 5 min followed by a 10 min incubation at room temperature prior to adding 8 μl of the reverse transcriptase reaction mixture. The reverse transcriptase mixture comprised the following: 2.5x First Strand Buffer (Invitrogen), 0.025 M DTT (Invitrogen), 0.5U/μl of RNaseOUT (Invitrogen) and 12.5U/μl of SuperScript-II enzyme (Invitrogen) all in nuclease-free water. The same mixture without SuperScript-II enzyme was used for the genomic DNA control samples. The reaction was conducted in a Biometra T-Professional thermocycler with the following program: 42 °C for 50 min followed by 70 °C for 15 min. The cDNA was stored at −20 °C until needed.

Agosto L.M., Hirnet J.B., Michaels D.H., Shaik-Dasthagirisaheb Y.B., Gibson F.C., Viglianti G, & Henderson A.J. (2016). Porphyromonas gingivalis-mediated signaling through TLR4 mediates persistent HIV infection of primary macrophages. Virology, 499, 72-81.

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Innate Immune Gene Expression in Larval Treatments

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We evaluated the gene expression of some innate immune genes in larvae exposed to different treatments (Table 1). Three pools of 5 larvae per treatment were analyzed. Each pool of larvae was homogenized with a 25-gauge needle and RNA was obtained with the SV Total RNA Isolation System (Promega). cDNAs were synthesized using the ImProm-II™ Reverse Transcription System (Promega) according to the manufacturer's instructions in a TProfessional Thermocycler (Biometra). qPCR was performed in the LightCycler96 (Roche) using FastStart Essential DNA Green Master (Roche) in a 10 μL reaction with a final primer concentration of 500 nM. The primer sequences are detailed in Table 1. The thermal profile used was 95°C 10 min, 40 × (95°C × 30 s, 60°C × 30 s, 72°C × 30 s). Relative expression of RNAm was calculated using 2^−ΔΔCT adjusted to primer efficiency (Pfaffl, 2001 (link)). beta actin 1 was used as housekeeping gene.

Caruffo M., Navarrete N.C., Salgado O.A., Faúndez N.B., Gajardo M.C., Feijóo C.G., Reyes-Jara A., García K, & Navarrete P. (2016). Protective Yeasts Control V. anguillarum Pathogenicity and Modulate the Innate Immune Response of Challenged Zebrafish (Danio rerio) Larvae. Frontiers in Cellular and Infection Microbiology, 6, 127.

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Quantitative HIV-1 Integration Assay

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A minimum input of 8x10⁵ cells per experimental condition was lysed in Tris-EDTA buffer prior to Alu-PCR. Nested PCR strategy was adapted from Agosto et al., 2007 [20 (link)]. Briefly, integrated HIV-1 DNA was amplified using forward primers for the luciferase sequence and reverse primers for human Alu (see S1 File). The first reaction was performed on a TProfessional Thermocycler from Biometra according to the following conditions: 4 m at 95° followed by 20 cycles of 15 s at 93°C, 15 s at 50°C, and 2.5 m at 70°C. A second round of amplification was then performed using a forward primer, a reverse primer, and a probe for real time PCR within the HIV-1 3’ R / U5 region (see S1 File). The amount of amplified copies of HIV-1 was determined based on an NL4-3 plasmid copy standard. The second reaction was performed on an Applied Biosystems QuantStudio 3 Real-Time PCR system with heating for 4 m at 95° and real-time PCR conditions of denaturation for 15 s at 95°C, annealing for 30 s at 60°C, and extension for 1 m at 72°C.

Gagne M., Michaels D., Schiralli Lester G.M., Gummuluru S., Wong W.W, & Henderson A.J. (2019). Strength of T cell signaling regulates HIV-1 replication and establishment of latency. PLoS Pathogens, 15(5), e1007802.

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Quantitative RT-PCR Analysis of Gene Expression

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Total RNA was isolated from cells by using TRIzol reagent (Takara Bio, Tokyo, Japan). On the basis of the instructions of the reverse transcriptase kit (Takara, Tokyo, Japan), cDNA was synthesized using 2 μg of the total RNA in TProfessional Thermocycler (Biometra, Berlin, Germany). Then, cDNA samples were subjected to qRT-PCR for 40 cycles by using TB Green™ Premix Ex Taq™ II (Takara, Tokyo, Japan) in Roche LightCycler 96 (Roche, Basel, Switzerland). Primers (Table 1) were designed with the approval of the Sango Biotech Co. Ltd. (Shanghai, China). GAPDH was used as an internal control. The results were calculated using the comparative cycle threshold (ΔΔCt) method.

Tang X., Sun Y., Xu C., Guo X., Sun J., Pan C, & Sun J. (2021). Caffeine Induces Autophagy and Apoptosis in Auditory Hair Cells via the SGK1/HIF-1α Pathway. Frontiers in Cell and Developmental Biology, 9, 751012.

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Reverse Transcription for RNA Analysis

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Total RNA (extracted and purified as described in Section 2.2.2 and Section 2.2.3) was reverse transcribed using a high-capacity cDNA reverse transcription kit. The sample was diluted to 100 ng/μL. We then mixed 10 μL RNA sample, 2 μL 10× RT Buffer, 0.8 μL 25× dNTP mix (100 mM), 2 μL 10X RT Random primer, 1 μL MultiScribe™ Reverse Transcriptase (50 U/μL), and 4.2 μL DEPC-treated ddH₂O. PCR was conducted using a thermocycler (TProfessional Thermocycler, Biometra, BM-070-801), and the reaction conditions were the following: 1. 25 °C for 10 min; 2. 37 °C for 120 min; 3. 85 °C for 5 min. The sample was diluted 50X and stored at −20 °C for later use.

Chen C.C., Huang C.W., Lin C.Y., Ho C.H., Pham H.N., Hsu T.H., Lin T.T., Chen R.H., Yang S.D., Chang C.I, & Gong H.Y. (2021). Development of Disease-Resistance-Associated Microsatellite DNA Markers for Selective Breeding of Tilapia (Oreochromis spp.) Farmed in Taiwan. Genes, 13(1), 99.

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ERIC-PCR Fingerprinting of R. leguminosarum

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For fingerprinting of R. leguminosarum bv. trifolii strain genomes by ERIC-PCR method, each DNA amplification reaction was set up in 5 µL mixture containing 1.650 µL of Multiplex PCR Master mix (Quiagen), 1.0 µL of RNase-free water (Quiagen), 0.350 µL of primer mixture (0.3 µM of each primer) and 2 µL of DNA (100 ng/μL). The ERIC-PCR DNA amplification with primers ERIC-1R (5′ ATGTAAGCTCCTGGGGATTCAC-3′) and ERIC-2 (5′-AAGTAAGTGACTGGGGTGAGCG-3′) (Versalovic et al. 1991 (link)) was carried out in TProfessional thermocycler (Biometra) with the following temperature profile: initial denaturation at 95 °C for 15 min, 30 cycles of denaturation at 90 °C for 30 s, annealing at 58 °C for 1 min, extension at 65 °C for 8 min, and final extension at 68 °C for 16 min (Versalovic et al. 1998 (link)).

Oleńska E, & Małek W. (2019). Genomic polymorphism of Trifolium repens root nodule symbionts from heavy metal-abundant 100-year-old waste heap in southern Poland. Archives of Microbiology, 201(10), 1405-1414.

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Quantifying TERT Gene Copy Number

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TERT gene copy number was quantified by Quantitative Multiplex Fluorescent Polymerase Chain Reaction (QMF-PCR) analysis. QMF-PCR was performed on a TProfessional thermocycler (Biometra, Archamps, France) as previously described [47 (link), 48 (link)]. Primers were designed for three fragments of the TERT gene on chromosome 5p (5’UTR, Exon 9 and 3’UTR) to cover the entire gene. The 5’UTR region is very GC-rich and was difficult to amplify. We therefore had to limit the analysis to exon 9 and 3’UTR. Seven genes on chromosomes 2p, 4p, 7q, 10q 11p, 11q (PVRL1, BOD1L, RET, ZNF638, AGBL2, CFTR and POR) were co-amplified as controls. Primers and technical details are described in Supplementary Methods. The fluorescence intensities of PCR products were correlated with the copy number of the relevant exons. Fourteen control DNAs were included in each experiment: 12 normal DNAs and 3 DNAs with known TERT copy number (loss, normal and gain, as determined by array-comparative genomic hybridization). A dosage quotient was calculated relative to all the other amplified exons in patients and controls. The range of ratios corresponding to two copies of TERT gene was set between 0.8 and 1.2 (mean +/- 3 standard deviations of values obtained in normal DNA samples). Ratios > 1.2 were considered as gains and < 0.8 as losses.

Gay-Bellile M., Véronèse L., Combes P., Eymard-Pierre E., Kwiatkowski F., Dauplat M.M., Cayre A., Privat M., Abrial C., Bignon Y.J., Mouret-Reynier M.A., Vago P., Penault-Llorca F, & Tchirkov A. (2017). TERT promoter status and gene copy number gains: effect on TERT expression and association with prognosis in breast cancer. Oncotarget, 8(44), 77540-77551.

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Polymerase Chain Reaction Protocol for PDAG Analysis

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A polymerase chain reaction (PCR) was performed in a TProfessional thermocycler (Biometra, Westburg, Leusden, the Netherlands) with labelled primers flanking the CAG stretch of the PDAGs (Biolegio, Nijmegen, the Netherlands; Supplementary Table 1). The PCR was performed using 10 ng of genomic DNA, 1 × OneTaq mastermix (New England Biolabs, Ipswich, MA, USA, OneTaq Hot start with GC Buffer mastermix), 1 μl of primer Mix A or B (Supplementary Table 1) and Aqua B. Braun water to a final volume of 10 μl. The PCR was run with 27 cycles of 30 s, denaturation at 94 °C, 1 min of annealing at 60 °C and 2 min elongation at 68 °C, preceded by 5 min of initial denaturation at 94 °C. Final elongation was performed at 69 °C for 5 min. Every PCR included a negative control without genomic DNA and a reference sample of CEPH 1347-02 genomic DNA. The PCR products were run on an ABI 3730 automatic DNA sequencer (Applied Biosystems, Foster City, CA, USA) and analysed using the GeneMarker software version 2.4.0. (SoftGenetics, State College, PA, USA). For every analysis, we included three controls with known CAG repeat lengths for each PDAG to assure that every run was performed reliably. All assessments were made with cases and controls randomized on plates and blinding with respect to disease status information.

Gardiner S.L., van Belzen M.J., Boogaard M.W., van Roon-Mom W.M., Rozing M.P., van Hemert A.M., Smit J.H., Beekman A.T., van Grootheest G., Schoevers R.A., Oude Voshaar R.C., Comijs H.C., Penninx B.W., van der Mast R.C., Roos R.A, & Aziz N.A. (2017). Large normal-range TBP and ATXN7 CAG repeat lengths are associated with increased lifetime risk of depression. Translational Psychiatry, 7(6), e1143-.

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