Dual flat block geneamp pcr system 9700

Manufactured by Thermo Fisher Scientific

The Dual Flat Block GeneAmp PCR System 9700 is a thermal cycler designed for polymerase chain reaction (PCR) amplification. It features two independently controlled heating blocks, allowing for simultaneous processing of multiple samples or different experimental setups.

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5 protocols using dual flat block geneamp pcr system 9700

Digital PCR Quantification of MYD88 L265P

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The digital PCR primer/probe mix of MYD88 L265P was obtained from Bio‐Rad (Refseq NC_000003.11, NG_023225.1, NT_022517.18, and NG_016954.1). For the detection and quantification of nucleic acids, the QuantStudio 3D PCR system (Thermo Fisher Scientific) was used. We mixed 7.5 μL of 2× Quant Studio 3D digital PCR Master Mix (Thermo Fisher Scientific), 0.75 μL of 20× target and wild probe (MYD88) (Bio‐Rad), and 10 ng of DNA to prepare a total volume of 15 μL. A 14.5 μL aliquot of the mixture was transferred to the Sample Loading Blade, and applied to the QuantStudio 3D Digital PCR 20K Chip using the Chip Loader. Amplification was performed using the Dual Flat Block Gene Amp PCR System 9700 (Thermo Fisher Scientific). In the analysis, the specific signal of the MYD88 L265P mutation was detected as a FAM signal, and the wild‐type signal was detected as a VIC signal. In this system, the fraction of the MYD88 L265P mutation dye signal over the total dye signal from the target gene (termed “Target/Total” within the QuantStudio™ 3D AnalysisSuite™ Software, Thermo Fisher Scientific) in the sample was calculated using the following formula:

Target / Total = \frac{(FAM) + (FAM + VIC)}{(FAM) + (FAM + VIC) + (VIC)} \times 100 [%]

The thresholds used were those automatically determined by the system and were readjusted manually only when the thresholds clearly crossed the cluster.

Yamagishi Y., Sasaki N., Nakano Y., Matushita Y., Omura T., Shimizu S., Saito K., Kobayashi K., Narita Y., Kondo A., Shiokawa Y., Nagane M, & Ichimura K. (2021). Liquid biopsy of cerebrospinal fluid for MYD88 L265P mutation is useful for diagnosis of central nervous system lymphoma. Cancer Science, 112(11), 4702-4710.

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EGFR Mutation Detection by PCR

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EGFR mutations were detected using a quantitative fluorescence PCR instrument (Cobas z480; Roche Diagnostics, Basel, Switzerland) and an EGFR detection kit (human EGFR gene mutations fluorescence PCR diagnostic kit; Amoy Diagnostics Co., Ltd., Xiamen, China) according to the manufacturers' protocols. EGFR mutations were also analyzed by digital PCR (dPCR; ABI-QuantStudio 3D, Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the protocols for the Dual Flat Block GeneAmp PCR System 9700 (Thermo Fisher Scientific, Inc.); dPCR primers and probes (cat no. A44177; Assay ID, HS000000026_rm; Thermo Fisher Scientific, Inc.) for EGFR mutation detection were purchased from Applied Biosystems. QuantStudio™ 3D AnalysisSuite™ software version 3.1.2 (Thermo Fisher Scientific, Inc.) was used for data acquisition and analysis.

Zhang Q., Nong J., Wang J., Yan Z., Yi L., Gao X., Liu Z., Zhang H, & Zhang S. (2019). Isolation of circulating tumor cells and detection of EGFR mutations in patients with non-small-cell lung cancer. Oncology Letters, 17(4), 3799-3807.

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Chip-based Digital PCR Analysis of miRNAs

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Chip-based dPCR was performed on a QuantStudio 3D Digital PCR System platform composed of the QuantStudio 3D Instrument (Thermo Fisher Scientific; 4489084), the Dual Flat Block GeneAmp PCR System 9700 (Thermo Fisher Scientific; 4484078), and the QuantStudio 3D Digital PCR Chip Loader (Thermo Fisher Scientific; 4482592). dPCR was performed according to the manufacturer’s instructions. In particular, enzyme activation was performed at 96 °C for 10 min, denaturation at 98 °C for 30 s, followed by annealing/extension at 56 °C for 2 min (40 cycles), and then final extension at 60 °C for 2 min. Analysis was executed with the online version of the QuantStudio 3D AnalysisSuite (Thermo Fisher Cloud, Waltham, MA, USA). The evaluated copy numbers for each miRNA are expressed as the mean of Log10 of the copy number/µL within a 95% confidence interval (CI). Upper and lower limits of the 95% CI are indicated within square brackets.
Primers and probes purchased from Thermo Fisher Scientific were labelled with FAM dyes and used to evaluate the expression of candidate miRNAs (Supplementary Table S1).

D’Alessandra Y., Valerio V., Moschetta D., Massaiu I., Bozzi M., Conte M., Parisi V., Ciccarelli M., Leosco D., Myasoedova V.A, & Poggio P. (2022). Extraction-Free Absolute Quantification of Circulating miRNAs by Chip-Based Digital PCR. Biomedicines, 10(6), 1354.

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Quantifying Proviral HIV-1 DNA in CD4+ T Cells

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Cellular DNA from memory CD4⁺ T cells from HIV-infected individuals was isolated and quantified as described above. Proviral loads were measured using the QuantStudio® 3D Digital PCR System (ThermoFisher). Briefly, 1500 ng of cellular DNA (per chip) was mixed with Quantstudio® 3D digital PCR mastermix and an HIV-1-specific TaqMan Detection Assay (Life Technologies). PCR mixes were loaded onto QuantStudio® 3D Digital PCR 20 K Chips v2 using a QuantStudio® 3D Digital PCR Chip Loader. Sealed chips were cycled using a Dual Flat BlockGeneAMP® PCR System 9700 (Applied Biosystems) at 96 °C for 10 min, followed by 41 cycles of 60 °C for 2 min and 98 °C for 30 s, with a final extension at 60 °C for 2 min. Fluorescence was measured with a QuantStudio 3D Digital PCR Instrument and analyzed using Analysis Suite dPCR Cloud Software (Thermo Fisher Connect™). The number of human cells represented in each PCR mix was based on the concentration of cellular DNA and mass of the human diploid genome (6.6 × 10⁻¹² g DNA/cell).

Joussef-Piña S., Nankya I., Nalukwago S., Baseke J., Rwambuya S., Winner D., Kyeyune F., Chervenak K., Thiel B., Asaad R., Dobrowolski C., Luttge B., Lawley B., Kityo C.M., Boom W.H., Karn J, & Quiñones-Mateu M.E. (2022). Reduced and highly diverse peripheral HIV-1 reservoir in virally suppressed patients infected with non-B HIV-1 strains in Uganda. Retrovirology, 19, 1.

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TaqMan Open Array Genotyping Protocol

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Open array was performed using the TaqMan^® assay (Applied Biosystems; Thermo Fisher Scientific, Inc.), and genotypes were captured using the OpenArray^® NT Genotyping system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Non-template control (process control) was used in each plate assay. DNA samples (50 ng/µl each sample) and TaqMan^® OpenArray Master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) were mixed in a 384 well plate and transferred to the OpenArray plate using an autoloader. The OpenArray plate was subsequently filled with immersion fluid, and sealed with glue. Multiplex TaqMan^® assay reactions were performed in a Dual Flat Block GeneAmp PCR system 9700 (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR cycling conditions were as follows: Initial denaturation at 93˚C for 10 min; followed by 50 cycles at 95˚C for 45 sec, 94˚C for 13 sec and 53˚C for 14 sec; and a final step at 25˚C for 2 min. Samples were stored at 4˚C until further use (11 (link),12 (link)).

Bautista-Medina M.A., Gallardo-Blanco H.L., Martinez-Garza L.E., Cerda-Flores R.M., Lavalle-Gonzalez F.J, & Villarreal-Perez J.Z. (2020). Association study in Mexican patients with thyrotoxic hypokalemic periodic paralysis. Biomedical Reports, 13(4), 24.

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