Taqman PCR primers q295 developed previously for X. fragariae detection [10 ] were used exclusively in this study. The 20 μL qPCR reaction contained 0.36 μM of forward and reverse primers, 0.17 μM of the Taqman probe, and 1x master mix from the DyNAmo Flash Probe qPCR Kit (Thermo Fisher Scientific, Pittsburgh, PA). The reactions were run in an Eppendorf Realplex4 master cycler at 95°C for 7 min, followed by 40 cycles of 95°C for 10 s and 55°C for 30 s. Non-template control was included in each run. Each sample was run in two technical replicates.
Dynamo flash probe qpcr kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The DyNAmo Flash Probe qPCR Kit is a real-time PCR reagent kit designed for quantitative analysis of DNA or RNA targets. The kit includes a ready-to-use master mix containing a modified Taq DNA polymerase, dNTPs, and buffer components optimized for probe-based qPCR. It is intended for use with probe-based detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Realplex4, by Eppendorf (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Applied biosystems viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Tri reagent solution, by Thermo Fisher Scientific (1 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Ribolock rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Steponeplus sequence detection system, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Lightcycler, by Roche (1 mentions)
6 protocols using dynamo flash probe qpcr kit
1
Taqman PCR Assay for X. fragariae
2
RNA Extraction and qPCR Analysis
3
Quantification of HBV DNA Markers
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Real-Time PCR amplification was performed using the DyNAmo Flash Probe qPCR Kit (Thermo Scientific, Life Science Research), in a 30 µL reaction mixture containing 5 µL of extracted DNA or standard plasmid pTHC dilutions. The final concentrations of the probes and primers were 0.1 µM and 0.5 µM, respectively.
The thermal cycling conditions were as follows: 10 minute at 95°C, followed by 45 cycles of 20 s at 95°C and 30 s at 62°C to amplify the HBV cccDNA and hTERT; and 8 minute at 95°C, followed by 50 cycles of 10 s at 95°C and 50 s at 56°C to amplify the HBV tDNA. The amounts of the HBV tDNA, cccDNA, and hTERT in each sample were determined by extrapolation from the external dilution curves of a multi-standard plasmid (pTHC) of known concentration, which incorporated all three templates in equimolar amounts. The results were expressed as the log10 HBV tDNA and cccDNA copies/106 cells, respectively.
The thermal cycling conditions were as follows: 10 minute at 95°C, followed by 45 cycles of 20 s at 95°C and 30 s at 62°C to amplify the HBV cccDNA and hTERT; and 8 minute at 95°C, followed by 50 cycles of 10 s at 95°C and 50 s at 56°C to amplify the HBV tDNA. The amounts of the HBV tDNA, cccDNA, and hTERT in each sample were determined by extrapolation from the external dilution curves of a multi-standard plasmid (pTHC) of known concentration, which incorporated all three templates in equimolar amounts. The results were expressed as the log10 HBV tDNA and cccDNA copies/106 cells, respectively.
4
Quantitative PCR of Immune Genes
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Total RNA was extracted from the cells using a TRI reagent solution (Invitrogen, Carlsbad, CA, USA). DNase I (Thermo Scientific) was used to remove potential genomic DNA contamination. Total RNA (6 μg) was digested in the presence of RNAse inhibitor (RiboLock RNase Inhibitor, Thermo Scientific) according to the manufacturer’s protocols. An aliquot of 1.5 μg of total RNA was used to make the first strand complementary cDNA (in 20 µL) using the Maxima First Strand cDNA Synthesis Kit (Thermo Scientific), according to the manufacturer’s protocol. Each PCR reaction (20 μL, three replicates in a 96-well plate) contained 10 μL DyNamo Flash Probe qPCR Kit (Thermo Scientific), 1.3 μL of cDNA, 1 μL of each primer (10 μM) and 6.7 μL of H2O. GAPDH-VIC (Hs02786624_g1), CXCL10-FAM (Hs00171042_m1), IFNγ-FAM (Hs00989291_m1) and IFNβ-FAM (Hs0027188_s1) were used for qPCR analysis (Thermo Fisher Scientific). qPCR was conducted using the Applied Biosystems ViiA 7 Real-Time PCR System (Thermo Fisher Scientific).
5
Quantification of Chemokine and Cathepsin mRNA
6
Sensitive Real-Time PCR Detection
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16S rRNA-and OspA-specific primers and probes, as described earlier (6, 22) . Real-time PCR with LightCycler ® (Roche, Basel, Switzerland) was also performed. Briefly, PCR reactions contained 10 µl 2×MasterMix of the DyNAmo Flash Probe qPCR Kit (ThermoFisher Scientific, Waltham, MA, USA), 1 µl 10 µM the corresponding forward and reverse primers, 0.5 µl 10 µM probe, and 100 ng template DNA in a total reaction volume of 20 µl. PCR cycling (60 cycles of 95°C 15 s, 60°C 1 min) was performed with a LightCycler instrument (Roche).
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