PCR was performed with 50 ng DNA on the FlexCycler (Analytik Jena, Jena, Germany) thermal cycler. Briefly, the reaction was carried out using the innuTaq HOT-A DNA Polymerase with 10× PCR buffer with KCl (Analytik Jena), in a total volume of 25 µl, containing 0.2 µm of each primer, 200 µm of each dNTP, 1.5 mM MgCl2, and 0.5 U DNA polymerase. An initial denaturation for 3 min at 94 °C was followed by 40 cycles of 94 °C for 45 s, 65 °C for 45 s, and 72 °C for 45 s, with a final elongation step of 10 min for 72 °C. Primers were obtained from Biomers (Ulm, Germany) and sequences were taken from the literature28 (link). Sequencing was performed with the GenomeLab DTCS Quick Start Kit (Beckman Coulter, Pasadena, CA) on the GenomeLab GeXP (Beckman Coulter).
Flexcycler
Manufactured by Analytik Jena
Sourced in Germany, United States
The FlexCycler is a thermal cycler designed for PCR amplification. It features a compact design and a color touchscreen interface. The FlexCycler can accommodate a range of sample block sizes to fit different experimental needs.
Automatically generated - may contain errors
Lab products found in correlation
Iscript cdna synthesis kit, by Bio-Rad (4 mentions)
Cfx manager, by Bio-Rad (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Gotaq qpcr master mix, by Promega (3 mentions)
25 bp dna ladder, by Promega (2 mentions)
Tprofessional thermocycler, by Analytik Jena (2 mentions)
Rest 2009, by Qiagen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Genomelab dtcs quick start kit, by Beckman Coulter (1 mentions)
Genomelab gexp, by Beckman Coulter (1 mentions)
Rneasy mini extraction kit, by Qiagen (1 mentions)
C1000 thermocycler, by Bio-Rad (1 mentions)
Ssofast evagreen supermix, by Bio-Rad (1 mentions)
Cfx manager 3, by Bio-Rad (1 mentions)
Primeprep genomic dna isolation kit from tissue, by GeNet Bio (1 mentions)
Taq polymerase, by Vivantis Technologies (1 mentions)
Relative expression software tool v 2.0.13, by Qiagen (1 mentions)
Exgene stool dna mini kit, by GeneAll (1 mentions)
Nanodrop, by Avantor (1 mentions)
18 protocols using flexcycler
1
PCR Amplification and Sequencing Protocol
2
Gene Expression Analysis of Bone Cells
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A RNeasy Mini Extraction Kit (Qiagen, Hilden, Germany) was used to extract the RNA. Complementary DNA (cDNA) synthesis was performed in a FlexCycler thermal cycler (Analytik-Jena, Jena, Germany) using the iScript cDNA synthesis kit (Bio-Rad, Munich, Germany) according to the manufacturer's instructions. Standards were prepared by a tenfold dilution series between 1 and 1 × 10−5. cDNA was stored at −20°C.
For qPCR, the SsoFast EvaGreen Supermix (Bio-Rad) and 1 μg of cDNA were processed in a Bio-Rad C1000 ThermoCycler running the CFX96 Real-Time System. Cycling conditions were 30 sec at 95°C, 40 cycles of 5 sec at 95°C, and 10 sec at 60°C. Finally, a melt-curve analysis with 0.5°C increments every 5 sec from 65°C to 95°C was performed.
The target genes were Alpl (alkaline phosphatase), Bglap (bone gamma-carboxyglutamate (gla) protein or osteocalcin), Fmod (fibromodulin), and Runx2 (runt-related transcription factor 2); reference genes were Actb (actin beta) and Hprt (hypoxanthine guanine phosphoribosyl transferase). Primers were obtained from Qiagen and Sigma-Aldrich (Table 1 ). The primer covered at least one exon-intron junction and the negative first-deviation plots of the melting curve revealed specificity. Target gene expression was assessed using CFX Manager 3.1 software (Bio-Rad) and normalized to the reference genes.
For qPCR, the SsoFast EvaGreen Supermix (Bio-Rad) and 1 μg of cDNA were processed in a Bio-Rad C1000 ThermoCycler running the CFX96 Real-Time System. Cycling conditions were 30 sec at 95°C, 40 cycles of 5 sec at 95°C, and 10 sec at 60°C. Finally, a melt-curve analysis with 0.5°C increments every 5 sec from 65°C to 95°C was performed.
The target genes were Alpl (alkaline phosphatase), Bglap (bone gamma-carboxyglutamate (gla) protein or osteocalcin), Fmod (fibromodulin), and Runx2 (runt-related transcription factor 2); reference genes were Actb (actin beta) and Hprt (hypoxanthine guanine phosphoribosyl transferase). Primers were obtained from Qiagen and Sigma-Aldrich (
3
DNA Extraction and ITS1 Amplification Protocol
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At first, all stored samples were washed twice with PBS to remove ethanol. The DNA extraction procedure was accomplished using PrimePrep genomic DNA isolation kit from tissue (GeNet Bio, South Korea) based on the manufacturer’s protocol and the genomic DNA was kept at -20°C for PCR reaction. The concentration of each DNA sample was measured by NanoDrop (Thermo, USA) evaluation at A260.In each sample, an Internal Transcribed Spacer 1 (ITS1) fragment was amplified by conventional PCR, using a specific primer pair:
BDI (5/-GTCCTAACAAGGTTTCCGTA-3/) for the 18S region.
4S (5/-TCTAGCGTTCGAA(G/A)TGTCGATG-3/) for the 5.8S region. A 25 μl PCR mixture was prepared for each sample, containing 12.5 μl of Master Mix (Ampliqon, Denmark), 3 μl of extracted DNA, ten pmol of each primer and 6.5 μl of sterilized water. The following PCR program was carried out in an automated thermo cycler (FlexCycler, Analytik Jena, Germany): an initial denaturation step at 95°C for 5 min, 30 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 45 sec, and extension at 72°C for 45 sec, followed by a final extension step at 72°C for 5 min. Finally, the PCR products were separated by 1.5 % agarose gel electrophoresis and displayed under ultraviolet (UV) illumination.
BDI (5/-GTCCTAACAAGGTTTCCGTA-3/) for the 18S region.
4S (5/-TCTAGCGTTCGAA(G/A)TGTCGATG-3/) for the 5.8S region. A 25 μl PCR mixture was prepared for each sample, containing 12.5 μl of Master Mix (Ampliqon, Denmark), 3 μl of extracted DNA, ten pmol of each primer and 6.5 μl of sterilized water. The following PCR program was carried out in an automated thermo cycler (FlexCycler, Analytik Jena, Germany): an initial denaturation step at 95°C for 5 min, 30 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 45 sec, and extension at 72°C for 45 sec, followed by a final extension step at 72°C for 5 min. Finally, the PCR products were separated by 1.5 % agarose gel electrophoresis and displayed under ultraviolet (UV) illumination.
4
Microsatellite Genotyping of Bactrocera
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Sets of seven (Bd1, Bd9, Bd15, Bd19, Bd39, Bd42, and Bd85B) and five (Bp58, Bp73, Bp125, Bp173, and Bp181) microsatellite loci - previously isolated and characterized from B. dorsalis s.s. [31 ] and B. papayae[18 ], respectively - were used (Additional file 1 : Table S1). Microsatellite DNA amplifications were set up in a 15- μl volume reaction containing 100 ng of genomic DNA, 1 × buffer, 2.5 mM MgCl2, 25 μM dNTPs, 0.5 U Taq polymerase (Vivantis) and 5 μM of each primer. PCRs were performed using a thermal cycler Flexcycler (AnalytikJena, Germany) using the following conditions: 5 min at 94°C, 29 cycles of 30 s at 94°C, 90 s at Ta of each primer pair (Additional file 1 : Table S1) and 90 s at 72°C, and an additional 5 min of elongation at 72°C at the end of the process. Electrophoresis and allele scoring were determined in 6% or 12% (w/v) non-denaturing polyacrylamide gel in 1xTBE buffer at 800 V for 6 or 10 hrs, respectively, stained with 0.5 mg/l ethidium bromide [32 ] and photographed under UV light. PCR product size was measured with a 25 bp DNA ladder (Promega, USA). For each locus analysis, samples with no PCR product were declared null allele (missing data), if two PCR attempts had been carried out.
5
Genotyping of SNPs Associated with Cardiovascular Disease
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Genomic DNA was extracted from blood samples of the patients and normal subjects using the standard salting out method. SNPs rs1333045, rs4977574, rs1333048 and rs10757278 were genotyped by tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) (14 (link)). PCR was performed in 25 µL total volume using Taq (2x) red master mix (Ampliqon, Denmark) and 0.5 µL of each forward and reverse primer (10 pmol) in a FlexCycler (Analytik Jena, Germany). The cycling conditions were an initial denaturation at 94˚C for 4 minutes, followed by 35 cycles of 94˚C for 45 seconds, annealing temperature for 45 seconds and 72˚C for 55 seconds, with a final extension of 72˚C for 5 minutes. Specific annealing temperatures were 45˚C for rs1333048, 53˚C for rs4977574, 52˚C for rs1333045 and 54˚C for rs10757278. The primers were designed by PRIMER1 (http://cedar. genetics.soton.ac.uk/public_html/primer1.html.) and are listed in Table 1.
6
Standardized PCR Protocol for Environmental DNA
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All PCR reactions were performed with the primer pair AGF-LSU-EnvS and contained 2.5 µL 10 × PCR buffer, 6 mM MgCl2 (50mM), 200 µM dNTPs (10 mM each), 250 nM of each primer, and 0.75 U PlatinumTM Taq DNA Polymerase (Life Technologies, Carlsbad, CA, USA), and 1 µL DNA template (concentrations between 0.6 and 17.5 ng/µL) in a total reaction volume of 25 µL. In no template controls (NTC), the DNA template was replaced by nuclease-free water. The PCR program consisted of 3 min initial denaturation/activation at 95 °C, 35 cycles of 20 s denaturation at 95 °C, 30 s annealing at 62 °C, and 60 s elongation at 72 °C. A final extension of 10 min at 72 °C concluded the PCR run with a hold at 4 °C thereafter. The PCR runs were performed on a TProfessional Thermocycler (Biometra, Jena, Germany) or a Flexcycler (Analytik Jena, Germany). Agarose gel electrophoresis (1% w/v agarose in TAE buffer, 3 µL PCR product, 60 min, voltage of 100 V, 2 µL SERVA DNA Stain G dye) was used to visualize the PCR products.
7
RNA Isolation and qRT-PCR Analysis
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Total RNA was isolated with the RNeasy Mini Kit (74106, QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. Reverse Transcription of mRNA was performed with iScript cDNA Synthesis Kit (170–8891, Bio-Rad, Hercules, California, USA) on FlexCycler (Analytik Jena AG, Jena, Germany). QIAGEN primers for human GAPDH (QT01192646), ACTA2 (QT00088102), COL1A1 (QT00037793), SNAI1 (xxxxxxxxxx), TLR5 (QT01009596), VIM (QT00095795) and primers for mouse Gapdh (QT01658692), Tlr5 (QT02328221), Acta2 (QT00140119), Col1a1 (QT00162204) were used with GoTaq qPCR Master Mix (Promega, Madison, USA) on RT-qPCR thermocycler CFX96 Real-Time System (Bio-Rad Laboratories, Hercules, California, USA). Results were analysed with the Bio-Rad CFX-Manager (Bio-Rad Laboratories) and normalised with GAPDH mRNA content for each sample. Raw data were further analysed with REST 2009 (Relative Expression Software Tool V.2.0.13., QIAGEN).
8
Detecting E. granulosus in Stool Samples
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Genomic DNA of all stool specimens were extracted, using ExGene Stool DNA mini kit (GeneAll, South Korea), according the manufacturer’s instructions. The primers, Eg1121a, 5′-GAATGCAAGCAGCAGATG-3′ (forward) and Eg1122a, 5′-GAGATGAGTGAGAAGGA GTG-3′ (reverse) were used for specific identification of E. granulosus sensu lato. This primer pair amplifies a 133-bp fragment of a repeat unit of the parasite genome with suitable sensitivity and specificity [20 (link),21 (link)]. The PCR was carried out in 25 μl reaction volumes in FlexCycler (Analytik Jena, Germany) machine. The thermal profile included an initial denaturation at 94°C for 5 min; followed by 35 cycles, each of 30 s at 94°C, 45 s at 50°C and 35 s at 72°C; and a final extension of 10 min at 72°C. The amplification products were subjected to electrophoresis on 2% agarose gel in TAE buffer.
9
Isolation and Characterization of a DBP-Degrading Bacterium
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A DBP degrading bacterium was isolated on solid medium using MSM agar supplemented with 5 µL DBP (distributed on the agar surface); as a control, plates without a carbon source were prepared. The 16S rRNA gene was directly amplified from bacterial suspension using the bacterial primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The 50 µL PCR reaction contained 25 µL Red Tag polymerase (Genaxxon bioscience, Ulm, Germany), 2.5 µL forward primer (10 µM stock solution), 2.5 µL reverse primers (10 µM stock solution), 20 µL sterile distilled water and a small amount of the pure colony. PCR mixture was applied to a temperature cycler (Flex cycler, Analytik Jena, Jena, Germany). The program used for PCR consisted of an initial denaturation step at 95 °C for 5 min, followed by 34 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s, plus a final step at 72 °C for 5 min. The PCR product was evaluated by 1% agarose gel electrophoresis, quantified by a NanoDrop (PeqLab, Erlangen, Germany), and sequenced by Sanger sequencing (Eurofins Genomics Germany, Ebersberg, Germany).
10
Genotyping Bactrocera Fruit Flies
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Thirty male individuals of each population (B. carambolae (Jakarta), B. carambolae (Sumatra), B. dorsalis (Nakhon Pathom), the Salaya1 colony, and the resulting introgressive strain (Salaya5)) were used for genotyping. The genomic DNA was extracted from each individual fly as described by Aketarawong and colleagues [45 (link)]. PCR amplifications using two sets of microsatellite loci; five polymorphic loci (Bd1, Bd9, Bp125, Bp173, and Bp181) and four Y-pseudo-linked markers (Bd15, Bd42, Bp58, and Bp73) [30 (link)] were carried out in order to assess general genetic background and validate the existence of the Y- Awp+ component in the Salaya5 strain, respectively. PCR reactions and conditions were performed using the thermal cycler Flexcycler (Analytik Jena AG, Jena, Germany). PCR products were run on 6% or 12% polyacrylamide gels and were scored in comparison with a 25 bp DNA ladder (Promega, Madison, WI, USA) as described in [13 (link)].
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