Turbo dna free dnase

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, Japan, France

Turbo DNA-free DNase is a lab equipment product designed to remove DNA contamination from RNA samples. It functions by digesting single-stranded and double-stranded DNA, leaving the RNA intact.

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Turbo DNase (2545 citations) DNase (1122 citations) Turbo DNA-free (359 citations) DNA-free (81 citations) TURBO DNA-free DNase kit (78 citations) DNA-free DNase (38 citations) Turbo DNase-free (18 citations) TURBO DNase (TURBO DNA-free Kit; (16 citations) Turbo DNA-free DNase I (15 citations) Turbo DNA-free DNase treatment (15 citations)

122 protocols using turbo dna free dnase

Quantifying Luciferase Expression in Transfected 293T Cells

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Two out of three replicates of transfected 293T cells were lysed with RLT buffer, and total RNA was extracted with RNeasy kit (Qiagen). To remove potential DNA contamination from plasmids used to transfect the cells, 5 μg of extracted RNA was incubated for 3 hours at 37°C with a mixture of 2 units Turbo DNA-free DNase (Applied Biosystems) and 2 units DpnI (NEB). Reverse transcription using High-fidelity Transcriptor Kit (Roche) was then performed with standardized amount of RNA using Oligo(dT)12. Synthesized cDNA was diluted 5 times. Real-time PCR of firefly and renilla luciferase genes were performed on a Roche 480 Lightcycler instrument using the SYBR Green I Master Mix (Roche), following the manufacturer’s instructions. In each reaction, 5 μL of cDNA, 10μL of the probe master mix (2×), 0.5 μM each of forward and reverse primers, and water was used. Plasmid DNA containing the firefly and renilla luciferase genes were used as standards diluted serially at 10-fold. The reaction has a hot start step at 95°C for 10 min for 1 cycle, 45 cycles of denaturation at 95°C for 10 s, annealing at 62 °C for 10 s, and extension at 72°C for 40 s.

Cheung P.P., Watson S.J., Choy K.T., Sia S.F., Wong D.D., Poon L.L., Kellam P., Guan Y., Peiris J.M, & Yen H.L. (2014). Generation and characterization of influenza A viruses with altered polymerase fidelity. Nature communications, 5, 4794.

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Quantifying Luciferase Expression in Transfected 293T Cells

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RNA Extraction and RT-qPCR Protocol

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RNA was extracted as described (Box et al. 2011 (link)), using phenol equilibrated to pH8, followed by lithium chloride precipitation. RNA was DNase-treated with Turbo DNA-Free DNase (Life Technologies). cDNA was synthesized with SuperScript III reverse transcriptase (Life Technologies) using either gene-specific primers or oligo dT (12–18). qPCR was performed using SYBR Green master mix II on a LightCycler 480 II (both Roche). Primer sequences are listed in Supplemental Table S2. UBC was used as the normalization gene control.

Qüesta J.I., Antoniou-Kourounioti R.L., Rosa S., Li P., Duncan S., Whittaker C., Howard M, & Dean C. (2020). Noncoding SNPs influence a distinct phase of Polycomb silencing to destabilize long-term epigenetic memory at Arabidopsis FLC. Genes & Development, 34(5-6), 446-461.

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Total RNA Isolation and cDNA Synthesis

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Total RNA isolation with the RNeasy Kit (Qiagen, Hilden, Germany), DNase digestion of purified RNA with Turbo DNA-free DNase (Life Technologies, Waltham, Massachusetts, U.S.) and cDNA synthesis with M-MLV Reverse Transcriptase (RT) RNase H minus (Promega, Fitchburg, Wisconsin, USA) were performed according to the manufacturer’s instructions. Turbo DNA-free digestion (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was performed with 500 ng to 1.5 µg total RNA as recommended. At least 300 ng DNase-digested RNA was reverse transcribed with M-MLV RT as +RT reactions versus equal amounts of RNA under the same conditions without M-MLV RT as –RT reactions. cDNA synthesis was performed with oligo(dT) primers (Thermo Fisher Scientific).

Zobel T., Bock C.T., Kühl U., Rohde M., Lassner D., Schultheiss H.P, & Schmidt-Lucke C. (2019). Telbivudine Reduces Parvovirus B19-Induced Apoptosis in Circulating Angiogenic Cells. Viruses, 11(3), 227.

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Quantifying Gene Expression via RT-qPCR

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RNA was extracted as described (Questa et al., 2016), using acidic phenol followed by lithium chloride precipitation. RNA was DNase treated with Turbo DNA Free DNase, then transcribed into cDNA with SuperScript Reverse Transcriptase IV (both Life Technologies) with the gene-specific reverse primers detailed below. qPCR was performed using SYBRGreen Master Mix II on a LightCycler 480 II (both Roche) with primer pairs: VIN3 qPCR 1F 5′-TGCTTGTGGATCGTCTTGTCA-3′ and VIN3 qPCR 1R 5′-TTCTCCAGCATCCGAGC AAG-3′, FLC F 5′-AGCCAAGAAGACCGAACTCA-3′ and FLC R 5′-TTTGTCCAGCAGGTGACATC-3′, UBC qPCR F 5′-CTGCGACTC AGGGAATCTTCTAA-3′ and UBC qPCR R 5′-TTGTGCCATTGAATTGAACCC-3′, PP2A F2 5′-ACTGCATCTAAAGACAGAGTTCC-3′ and PP2A R2 5′-CCAAGCATGGCCGTATCATGT-3′. Results were normalized to the geometric mean of two standard genes, PP2A (At1g13320) and UBC (At5g25760).

Fiedler M., Franco-Echevarría E., Schulten A., Nielsen M., Rutherford T.J., Yeates A., Ahsan B., Dean C, & Bienz M. (2022). Head-to-tail polymerization by VEL proteins underpins cold-induced Polycomb silencing in flowering control. Cell Reports, 41(6), 111607.

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Adrenomedullin Receptor Expression in Lung Fibrosis

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These experiments were performed to determine whether lung uptake of PulmoBind, a specific adrenomedullin receptor ligand, was associated with reduce expression of this receptor in the lungs of rats with bleomycin-induced fibrosis. RNA was extracted using the Qiagen TRIzol reagent (Fisher Scientific) and treated with TURBO DNA-free DNase (Fisher Scientific). Extracted RNA was converted to cDNA using GoScript Reverse Transcriptase with 500–1000 ng starting material. The quality of total RNA was monitored by capillary electrophoresis (Experion, Biorad, ON, Canada). Quantitative real-time PCR (qPCR) was performed on an AB-7900HT real-time cycler using TaqMan gene expression assays (Life Technologies). Primers for RAMP2 and CLR (rat) were obtained from Life Technologies. qPCR data was analyzed using the ΔΔCt method, using Ubiquitin C (UBC) or GAPDH as normalization controls for lung tissue.

Harel F., Nguyen Q.T., Nsaibia M.J., Finnerty V., Morgan A., Sirois M., Villeneuve L., Calderone A., Bergeron A., Brochiero E., Tardif J.C., Shi Y, & Dupuis J. (2021). SPECT imaging of pulmonary vascular disease in bleomycin-induced lung fibrosis using a vascular endothelium tracer. Respiratory Research, 22, 240.

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Quantitative RT-PCR Analysis of RAMP2 and CLR

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RNA was extracted from homogenized lung tissue using the Qiagen TRIzol reagent (Fisher Scientific) and treated with TURBO DNA-free DNase (Fisher Scientific) as per manufacturer’s instructions. Extracted RNA was converted to cDNA using GoScript Reverse Transcriptase with 500–1000 ng starting material per reaction. The quality of total RNA was monitored by capillary electrophoresis (Experion, Biorad, ON, Canada). Quantitative real-time PCR (qPCR) was performed with QuantiTect SYBR Green PCR kit (from Qiagen, CA) on the Mx3005P real-time PCR machine (Stratagene, CA). Primers for RAMP2 and CLR (rat) were obtained from Qiagen (ON, Canada). PCR data was analyzed using software MxPro (Stratagene, CA). Comparative quantitative analysis was performed based on a delta-delta Ct (ΔΔCt) method using HPRT1 gene (Life technologies/Thermo Fisher Scientific,ON, Canada) as normalization controls.

Merabet N., Nsaibia M.J., Nguyen Q.T., Shi Y.F., Letourneau M., Fournier A., Tardif J.C., Harel F, & Dupuis J. (2019). PulmoBind Imaging Measures Reduction of Vascular Adrenomedullin Receptor Activity with Lack of effect of Sildenafil in Pulmonary Hypertension. Scientific Reports, 9, 6609.

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Quantitative Gene Expression Analysis of Imprinted Genes

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Total RNA was extracted from leucocytes using TRIZOL reagent (Invitrogen, USA), according to the manufacturer's instructions. RNA samples were treated with TURBO DNA-free

^{TM}

DNase (Ambion, Life Technologies, USA). A no-reverse transcription (no-RT) control was used for each sample to exclude any potential non-specific amplification of genomic DNA. The SuperScript

^{TM}

First-Strand Synthesis System (Invitrogen, USA) was used to reverse transcribe total RNA with random hexamer primers. Quantitative Real-Time PCR employed TaqMan

^{TM}

Gene Expression MasterMix reagent (Thermo Scientific, USA) and TaqMan

^{TM}

Gene Expression Assays for evaluation of Beta-2 Microglobulin (B2M, endogenous control) IGF2, MEST, and PEG10 transcription. The primers for Oct4B1 were previously described (Table I). Quantification of the results was performed using the 2

^{- Δ Δ Ct}

method after normalization against B2M.

Argyraki M., Katafigiotis S., Vavilis T., Papadopoulou Z., Tzimagiorgis G., Haidich A.B., Chatzimeletiou K., Grimbizis G., Tarlatzis B., Syrrou M, & Lambropoulos A. (2021). Influence of conception and delivery mode on stress response marker Oct4B1 and imprinted gene expression related to embryo development: A cohort study. International Journal of Reproductive Biomedicine, 19(3), 217-226.

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Quantifying HBV Envelope in Murine Liver

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RNA was extracted from frozen livers (left lobe) using ReliaPrep RNA Tissue Miniprep System (Promega, Z6111), according to the manufacturer’s instructions, and genomic DNA was removed using Ambion TURBO DNA-freeTM DNase (AM1907). DNA lysis was performed with 5 µg of extracted RNA. 200 ng of total RNA was reverse-transcribed with Superscript IV Vilo (Life Technologies, 11766050) before qPCR analysis for HBV envelope. Reactions were run and analyzed on Quant Studio 5 instrument (Life Technologies). Analysis were normalized to the reference gene Gapdh (Life Technologies, Mm99999915_g1). All experiments were performed in duplicate.

Fumagalli V., Di Lucia P., Venzin V., Bono E.B., Jordan R., Frey C.R., Delaney W., Chisari F.V., Guidotti L.G, & Iannacone M. (2020). Serum HBsAg clearance has minimal impact on CD8+ T cell responses in mouse models of HBV infection. The Journal of Experimental Medicine, 217(11), e20200298.

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Poplar RNA Extraction and Quantitative RT-PCR

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Total RNA was isolated from stems of two‐month‐old poplar plants using the RNeasy Mini Kit according to the vendor’s manual (Qiagen, http://www.qiagen.com). RNA was quantified and treated with TURBO DNA‐free^TM DNase (Ambion RNA by Life Technologies, http://www.invitrogen.com). First‐strand cDNA was synthesized from 1 µg of total RNA with the High‐Capacity cDNA reverse transcription kit followed by reverse transcription, according to the vendor’s manual from Applied Biosystems (ThermoFisher, http://www.thermofisher.com). Quantitative RT‐PCR was performed using the StepOne Plus Real‐Time PCR System from Applied Biosystems (ThermoFisher, http://www.thermofisher.com) and Fast Sybr Green Mix from Applied Biosystems (ThermoFisher, http://www.thermofisher.com). The transgene‐specific primers used were as follows: RGIL6 F‐CAA AAC ATT ACC ATC ACG CCA, and RGIL6 R‐GTA CAC CGA TTT CCC ACA ATG. House‐keeping gene (UBQ2) primers used were: UBQ2‐F: TCC AAT GGA ACG GCC ATT AA and UBQ2‐R: TGT ACT CTT TTG AAG TTG GTG T.

Yang H., Benatti M.R., Karve R.A., Fox A., Meilan R., Carpita N.C, & McCann M.C. (2019). Rhamnogalacturonan‐I is a determinant of cell–cell adhesion in poplar wood. Plant Biotechnology Journal, 18(4), 1027-1040.

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