PCR amplifications of the B2L gene of PPV and L1 ORF of PV were performed with primers shown in Table 1 in a total reaction volume of 25 μl as described by Inoshima et al. [21 (link)] and Forslund et al. [54 ], respectively. For the amplification of the CMLV HA gene PCR reaction was performed in a final volume of 25 μl that contained 1× PCR buffer (Vivantis Technologies, Malaysia), 10 mM dNTPs mix, 0.4 μM of each primer (HA-F and HA-R), one unit Taq DNA polymerase (Vivantis Technologies, Malaysia) and one μl DNA template. The PCR amplification was carried out in a Tpersonal Thermocycler (BIOMETRA, Germany) under the following conditions: initial denaturation at 94 °C for three min followed by 35 cycles each included denaturation step at 94 °C for 30 s, annealing step at 56 °C for 30 s and extension step at 72 °C for 30 s. A final extension step at 72 °C for seven min was included. PCR products were resolved by electrophoresis in 1.5 % agarose in TAE buffer (40 mM Tris–acetate pH 8.0,1 mM EDTA) and the gel, stained with ethidium bromide and photographed using ultraviolet gel documentation system (BIOMETRA, Germany).
Tpersonal thermocycler
Manufactured by Analytik Jena
Sourced in Germany
The TPersonal Thermocycler is a compact and versatile instrument designed for efficient DNA amplification. It features a rapid temperature transition rate and precise temperature control to ensure reliable and consistent PCR results. The TPersonal Thermocycler is a core tool for molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Pfu turbo dna polymerase, by Agilent Technologies (2 mentions)
Sybr premix ex taq kit, by Takara Bio (2 mentions)
Primescript rt reagent kit with mirnas specific rt primers, by Thermo Fisher Scientific (2 mentions)
20 μl reaction system, by Thermo Fisher Scientific (2 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression assay probe, by Thermo Fisher Scientific (2 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Optitaq polymerase, by EURx (2 mentions)
Q solution, by Qiagen (2 mentions)
Clean up kit, by A&A Biotechnology (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Pcr buffer, by Vivantis Technologies (1 mentions)
Taq dna polymerase, by Vivantis Technologies (1 mentions)
Metal affinity chromatography, by Qiagen (1 mentions)
Greensafe, by NZYTech (1 mentions)
Taq polymerase enzyme, by Promega (1 mentions)
27 protocols using tpersonal thermocycler
1
PCR amplification of viral genes
2
DNA Origami Folding and Purification
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The origamis were designed with the program cadnano (58 (link)), and their design confirmed with the software cando (32 (link), 59 (link)). Typical folding mixtures contained 20 nM scaffold, a 5× excess of staple strands (100 nM) and activator/inhibitor strands, and 22 mM MgCl2 for cylinders and 5 mM MgCl2 for cuboids (43 (link)).
DNA origamis were folded in a Biometra TPersonal Thermocycler.
Temperature ramp for annealing: 80°C: 15 min, 80° to 60°C: 5 min per 1°C, 60° to 40°C: 3 hours per 1°C, 40° to 25°C: 1 hour per 1°C, stay at 4°C, Lid temp: 80°C.
Folded DNA origamis were stored at 4°C until further use. The folded origami mixtures were purified by spin filtration with Amicon 100-kDa spin filters at 10,000g and 15°C for 5 min. The samples were washed six times with FoB5 buffer [5 mM tris, 1 mM EDTA, 5 mM NaCl, and 5 mM MgCl2 (pH 7.2)] (60 (link)) and recovered by turning the filter upside down into a fresh tube and centrifuging at 5000g for 3 min.
DNA origamis were folded in a Biometra TPersonal Thermocycler.
Temperature ramp for annealing: 80°C: 15 min, 80° to 60°C: 5 min per 1°C, 60° to 40°C: 3 hours per 1°C, 40° to 25°C: 1 hour per 1°C, stay at 4°C, Lid temp: 80°C.
Folded DNA origamis were stored at 4°C until further use. The folded origami mixtures were purified by spin filtration with Amicon 100-kDa spin filters at 10,000g and 15°C for 5 min. The samples were washed six times with FoB5 buffer [5 mM tris, 1 mM EDTA, 5 mM NaCl, and 5 mM MgCl2 (pH 7.2)] (60 (link)) and recovered by turning the filter upside down into a fresh tube and centrifuging at 5000g for 3 min.
3
Recombinant Proteins Expression and Purification
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The full-length coding sequences of gTrxR and gEF1Bγ (GiardiaDB accession number GL50803_9827 and GL50803_12102, respectively) were PCR amplified from the Giardia WB-C6 genomic DNA using primers reported in Supplemental Table S1 . PCRs were performed on a T-Personal Thermocycler (Biometra, Göttingen, Germany) using 100 ng of gDNA, 10 units of high fidelity Pfu turbo DNA polymerase (Agilent Technologies, Santa Clara, CA, USA), 50 μM dNTP, 20 pmol of each primer in 50 μl of reaction mixture. Amplification conditions were: 1 cycle at 95 °C for 2 min; 30 cycles at 95 °C for 30 s, 56 °C for 30 s and 72 °C for 30 s; and 1 cycle at 72 °C for 7 min. The coding sequence of g14-3-3 was excised from p14-X vector3. For the expression of N-terminal 6xHIS-tagged fusion protein in Escherichia coli, the BamHI/NotI digested fragments were cloned in BamHI/NotI linearized pQ30 vector (Qiagen, Germany) and transformed in M15 strain. Expression of recombinant proteins and purification under native conditions by metal affinity chromatography (Qiagen, Germany) were performed as described (Lalle et al., 2015 (link)).
4
Multiplex PCR Amplification of Bacterial Genes
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For PCR amplification, bacterial cells were harvested from a fresh overnight culture on blood agar plates, and a dense suspension of cells was dissolved in 100 µL of ddH2O. Multiplex PCR amplifications were carried out with the primers GHDE-F-7m (0.5 µM), GE-R-32m (0.8 µM), SER3-F-1 (0.1 µM), SER3-R-108 (0.1 µM), GE-R-34, GLF-F-151, and GE-R-38 (Table S4 ). The reactions mix was carried out in a volume of 50 µL, with 0.5 units of HotMaster Taq Polymerase (5PRIME, VWR International Eurolab, Barcelona, Spain), buffer 1×, MgCl2 1.5 mM, dNTPs 10 mM each, and 5 µL of cell suspensions. PCR was undertaken in a T-personal Thermocycler (Biometra, Göttingen, Germany) with initial denaturation at 94 °C for 5 s, followed by 30 cycles of 94 °C for 5 s, 48 °C for 20 s and 65 °C for 10 min, and a final extension at 65 °C for 10 min.
5
Genotyping SLCO1B1 c.521T>C Variant
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The specific polymorphic variant of the SLCO1B1 gene, the c.521 T > C SNP analyzed in this study (GenBank accession no. NC_000012.10), was performed by a polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) assay. PCR reaction volume was 25µL containing 1 µM of each assay-specific primers, 0.3 mM deoxynucleotide triphosphate (dNTPs) (Promega), 3 mM MgCl2 (Promega), 1.5 U Taq polymerase enzyme (Promega), 1 × PCR GoTaq Buffer Mix, water, and ≈ 1 µg genomic DNA. The PCR included 40 cycles at 94 °C for denaturation of the genomic DNA and activation of the Taq polymerase enzyme, 55 °C for annealing of the primers, and 72 °C for extension.
The PCR assay was performed using Tpersonal Thermocycler (Biometra), and finally, electrophoretic separation on 2% (W/V) agarose gel, with a running time of 90 min at 80 V in 1X TAE buffer (Tris–acetate-EDTA buffer; 40 mM Tris, 20 mM acetate, 1 mM EDTA), and visualization of the gel-separated PCR products with Green Safe (NZYTech) staining under UV light (AlphaImager, AlphaInnotech).
The PCR assay was performed using Tpersonal Thermocycler (Biometra), and finally, electrophoretic separation on 2% (W/V) agarose gel, with a running time of 90 min at 80 V in 1X TAE buffer (Tris–acetate-EDTA buffer; 40 mM Tris, 20 mM acetate, 1 mM EDTA), and visualization of the gel-separated PCR products with Green Safe (NZYTech) staining under UV light (AlphaImager, AlphaInnotech).
6
Molecular Identification of Dromedary Tick
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PCR amplification of ITS2 of the rRNA gene and two mitochondrial genes, COI and 16S rRNA, was performed, using both specifically designed primers and previously published primers (Table-1 ) [18 (link)-20 (link)]. H. dromedarii DNA (2 μl) and 0.3 μl of each primer (10 pmol) (Macrogen, Seoul, South Korea) were added to the PCR mixture, containing one unit of Max Taq DNA Polymerase (Vivantis Technologies, Malaysia), 5 μl 10× ViBuffer (Vivantis Technologies, Malaysia), and 2 μl dNTPs (10 mM). The volume was adjusted to 25 μl by adding distilled water. Thermal cycling was performed on a TPersonal Thermocycler (BIOMETRA, Germany) with an initial 15 min cycle at 95°C, followed by 35 cycles consisting of 30 s at 94°C, 1 min at 55°C or 60°C depending on the primers, and 1 min at 72°C, followed by a final 10 min extension step at 72°C. To rule out DNA or amplicon contamination, Molecular Grade Water was used as a negative control throughout each step of the protocol. The PCR amplicons were sequenced by Macrogen Inc. (Seoul, South Korea) using BigDye (Applied Biosystems, California, USA) on an ABI3730XL DNA sequencer (Applied Biosystems, California, USA).
7
Quantitative miRNA and mRNA Analysis
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The total RNA from cells was extracted with the mirVana miRNA isolation kit (Ambion, USA) according to the manufacturer’s protocol. The cDNA was synthesized from total RNA using PrimeScript™ RT Reagent Kit with miRNAs specific RT primers (Applied Biosystems, Waltham, MA) (Takara, Dalian, China) in a total reaction volume of 10 μl in TPersonal Thermocycler (Biometra, Göttingen, Germany) by following the manufacturer’s instructions. Then, miRNA cDNA was quantified using SYBR Premix Ex Taq kit (Takara, Dalian, China) in a 20-μl reaction system (Applied Biosystems, Foster city, CA). Expression data were uniformly normalized to U6, serving as the internal control. For mRNA dosage studies, complementary DNA was obtained with PrimeScript RT reagent Kit (Takara, Dalian, China) and then used as template to quantify DNMT1, DNMT3A, DNMT3B, and GRIA3 levels by SYBR Green RT-PCR Kit (Takara, Dalian, China). The relative expression levels were evaluated by using the 2−∆∆Ct method.
8
Quantifying Gene Expression in Tight Junctions and Inflammation
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Cells were collected and stored as cell pellets. Total RNA was isolated using the RNeasy® Mini kit (Qiagen, Germany) as per the manufacturer’s instructions. 1000 ng RNA was reverse transcribed to produce cDNA (High-Capacity cDNA Reverse Transcription Kit; Applied Biosystems, USA) at 25 °C for 10 min, 37 °C for 30 min and then 85 °C for 5 min, using a T-Personal Thermocycler (Biometra, Germany). qPCR was carried out to quantify gene expression of tight junction proteins (ZO-1: Hs01551861_m1, claudin-5: Hs01561351_m1, occludin: Hs01561351_m1), Nitric Oxide Synthase (NOS) (inducible NOS: Hs01075529_m1, endothelial NOS: Hs01574665_m1) and pro-inflammatory genes (IL-1β: Hs01555410_m1, IL-6: Hs00174131_m1, IL-8: Hs00174103_m1, COX-2: Hs00153133_m1, TNFα: Hs00174128_m1, Nuclear Factor Kappa B (NFκB): Hs00765730_m1, p65: Hs01042014_m1). TaqMan Universal PCR Master Mix (Applied Biosystems, USA), with TaqMan Gene Expression Assay Probes obtained from Applied Biosystems (USA), was used for qPCR. The housekeeping gene used was β-Actin (Hs99999903_m1). Analysis was done using the 7500 Real-time PCR System (Applied Biosystems, USA). The PCR reaction conditions were as follows: 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The comparative CT (ΔΔCT) method was used to determine the relative mRNA expression of genes of interest.
9
Piroplasms 18S rRNA Gene Amplification
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A 450 bp long portion of the 18S rRNA gene of piroplasms was amplified with the primers PIRO-A1 [13] (link) 5′-AGG GAG CCT GAG AGA CGG CTA CC-3′ and PIRO-B [14] (link)
5′-TTA AAT ACG AAT GCC CCC AAC-3′ . The reaction mixture contained 1×concentration of Coralload PCR Buffer, 1.5 mM of MgCl2, 0.2 mM of each dNTP, 25 pmol of each primer and 1 U of HotStarTaq Plus DNA Polymerase (QIAgen GmbH, Hilden, Germany) in a final volume of 25 µl. The reaction was run in a T-personal thermocycler (Biometra GmbH, Göttingen, Germany) according to the following program: initial denaturation for 5 min at 95°C was followed by a cycle 94°C for 30 s, 60°C for 30 s and 72°C for 40 s, repeated 35 times and finished with 72°C for 10 min. PCR products were visualized in 1.5% agarose gel prestained with ethidium-bromide. Strongly positive samples were selected for sequencing done by the Macrogen Inc. (Seoul, South Korea). Representative sequences were submitted to the GenBank (accession numbers KJ941104-12). In all PCR procedures positive (Babesia canis DNA) and negative controls (sterile deionized water) were included.
10
Multimodal Characterization of DNA Origami
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