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For general laboratory use.
Please centrifuge briefly before opening (volume ≤2 ml).Shipping: shipped on gel packs
Storage Conditions: store dark at -20 °C
avoid freeze/thaw cycles, store dark
stable at 4 °C for up to 4 weeksShelf Life: 12 months
Form: liquid
Concentration: 2x
Description:
SCRIPT Direct RT-qPCR GreenMaster is designed for quantitative real-time analysis of target RNA directly from animal- or plant tissue, swabs and blood. The mix allows robust amplification avoiding the requirement of any prior RNA purification procedures based on a green-fluorescent DNA stain that is structurally similar to SYBR® GREEN.
It allows fast and easy quantification of sample RNA over a wide dynamic range with exceptional sensitivity and precision.
The mix contains all reagents required for RT-qPCR (except template and primers) in a premixed 2 x concentrated ready-to-use solution. High robustness, reliability and sensitivity of the mix are based on a genetically engineered reverse transcriptase and an antibody-blocked hot start polymerase in combination with an optimized and well-balanced buffer system.
The mix ensures fast and easy preparation with a minimum of pipetting steps and is specially recommended for:- Direct amplification of target RNA from various tissues samples
- Direct detection of viral or bacterial RNA in nasal or throat swabs
- Direct PCR from whole samples
- Point-of-Care diagnostics.
Green-fluorescent DNA stain
Green-fluorescent DNA stain is structurally similar to SYBR® GREEN and a superior DNA intercalator dye specially developed for DNA analysis applications including real-time PCR (qPCR). Upon binding to DNA, the non-fluorescent dye becomes highly fluorescent while showing no detectable inhibition to the PCR process. The dye is extremely stable, providing convenience during routine handling.
Green-fluorescent DNA stain is not recommended for high-resolution melting curve analysis (HRM).
To perform the assay, simply select the optical setting for SYBR® GREEN on the detection instrument.ROX Reference Dye
The mix can also be used in combination with ROX reference dye (#PCR-351) in PCR instruments that are compatible with the evaluation of the ROX signal.Content:
SCRIPT Direct RT-qPCR GreenMaster
2x conc. mix of Reverse Transcriptase, antibody-blocked Hot Start polymerase, dNTPs, green-fluorescent DNA stain, reaction buffer, additives and stabilizersDirect Extraction Buffer
5x conc.
Please handle with care and wear personal protective equipment!PCR-grade Water
Procedure
Before starting, take reagents out from fridge and allow to thaw completely. Vortex all reagents briefly and spin down the liquids.1. Sample preparation
1.a Blood Samples / Liquid Samples- Dilute 5x Direct Extraction Buffer to 1x concentrated Buffer with PCR-grade water.
- Transfer 2 μl of the Blood/Liquid Sample into a tube containing 100 μl to 200 μl of 1x concentrated Direct Extraction Buffer (a dilution of Blood 1:50 to 1:100 in 1x Direct Extraction Buffer is recommended).
- Close the tube and vortex for 15 sec
- Incubate the tube at room temperature (20-25 °C) for 2-3 min.
- Transfer 1-2 μl of the supernatant into a 20 μl RT-qPCR assay or 2-5 μl into a 50 μl RT-qPCR assay (see table for Preparation of the RT-qPCR Assay below).
1.b Samples from nasal or throat swabs
- Dilute 5x Direct Extraction Buffer to 1x concentrated Buffer with PCR-grade water.
- Transfer 200 μl 1x Direct Extraction Buffer into a 1.5 ml microtube
- Cut off the cotton tip with the collected nasal or throat swab and place it in the micro tube
- Close the tube and vortex for 15 sec
- Incubate at room temperature (20-25 °C) for 2-3 min
- Remove the cotton tip and squeeze it out at the rim of the tube
- Centrifuge briefly and transfer 1-2 μl of the supernatant into a 20 μl RT-qPCR assay or 2-5 μl into a 50 μl RT-qPCR assay (see table for Preparation of the RT-qPCR Assay below).
1.c Samples from Animal or Plant Tissue
- Dilute 5x Direct Extraction Buffer to 1x concentrated Buffer with PCR-grade water.
- Prepare a small piece from animal or plant tissue not exceeding 8 mm in diameter
- Crack plant seeds to less than 1 mm in diameter using a BeadBeater, Tissue Lyser or small hammer
- Place the sample in a 1.5 ml microtube
- Add 1x concentrated Direct Extraction Buffer to the tissue sample as following:
Sample size (diameter) 1-2 mm 3-4 mm 5-8 mm 1x Direct Extraction Buffer 50 μl 100 μl 200 μl - Mix briefly by tapping or vortexing and make sure that the sample is soaked with Direct Extraction Buffer
- Incubate at room temperature (20-25 °C) for 3 min
- Centrifuge briefly and transfer 1-2 μl of the supernatant into a 20 μl RT-qPCR assay or 2-5 μl into a 50 μl RT-qPCR assay (see table for Preparation of the RT-qPCR Assay below).
2. Preparation of the RT-qPCR Assay
Preparation of a master mix is crucial in quantitative PCR reactions to reduce pipetting errors. Prepare a master mix of all components except template as specified below. A reaction volume of 20-50 μl is recommended for most real-time instruments. Pipet with sterile filter tips and minimize the exposure of the labeled DNA probe to light. Perform the setup in an area separate from DNA preparation or analysis. No-template controls should be included in all amplifications.component stock conc. final conc. 20 μl
assay50 μl
assaySCRIPT Direct RT-qPCR GreenMaster 2x 1x 10 μl 25 μl Extracted Sample - - 1-2 μl 2-5 μl Forward Primer 11) 10 μM 300 nM 0.6 μl 1.5 μl Reverse Primer 11) 10 μM 300 nM 0.6 μl 1.5 μl ROX Reference Dye
#PCR-351 2)25 μM 500 nM 0.4 μl 1 μl PCR-grade water - - fill up to
20 μlfill up to
50 μl1) The optimal concentration for primers and probe may vary from 100 to 500 nM and should be optimized for each new assay set-up
2) The mix can optionally be used in combination with ROX reference dyeMix the tubes briefly and spin down to remove bubbles.
3. RT-PCR Cycling
Switch on the real-time PCR cycler and set all cycling parameters as recommended in the table below. Place the vials into the instrument and start the program.Reverse
transcription3)50-55 °C 10-15 min 1x Initial
denaturation95 °C 5 min 1x Denaturation
Annealing
Elongation95 °C
60-65 °C 4)
72 °C15 sec
20-30 sec
30-60 sec5)
35-45x3) A reverse transcription time of 10 min is recommended for optimal amplicon lengths between 100 and 200 bp. Longer amplicons up to 500 bp may require a prolonged incubation of 15 min. Add 3 min for each additional 100 bp. The optimal temperature depends on the structural features of the RNA. Increase the temperature to 55°C for difficult templates with high secondary structure. Note that optimal reaction time and temperature should be adjusted for each particular RNA.
4) The annealing temperature depends on the melting temperature of the primers.
5) The elongation time depends on the length of the amplicon. A time of 30 sec is sufficient for fragments < 500 bp.To obtain optimal specificity and amplification results an individual optimization of the recommended parameters is recommended for each particular sample/primer pair.
4. Data Analysis
- Calculate ct-values and evaluate the data according to the instruction of the cycler and requirements of the experiment/application.
® SYBR is a trademark of Molecular Probes, Inc.
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产品组分
内容 型号
规格 储存温度
绿色预混直接RT-qPCR试剂盒
PCR532-01 250次x20ul(2x1.25ml)
-20°C 绿色预混直接RT-qPCR试剂盒
PCR532-02 1250次x20ul(10x1.25ml)
-20°C 操作手册
1 1 常温
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营销中心
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注意事项
保存建议 厂家推荐蓝冰运输。当您收到产品后，按照说明书建议保存于-20°C。 -
FAQ
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Selected References:
Quan et al. (2009) Circular polymerase extension cloning of complex gene libraries and pathways. PLoS One. 4:e6441.Unveiling the Petunia hybrida Virome: Metatranscriptomic Profiling from the Bulgarian Market and In Vitro Cultures
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Journal of Virology | 2023 Sep 30 | 37695056
Cryptic splicing events result in unexpected protein products from calpain-10 (CAPN10) cDNA.
Yasuko Ono, Naoko Doi, ..., Ana María Salazar
Biochimica et biophysica acta. Molecular c… | 2022 Feb 18 | 34906616
Enzyme catalysis prior to aromatic residues: Reverse engineering of a dephospho-CoA kinase.
Mikhail Makarov, Jingwei Meng, ..., Klára Hlouchová
Protein science : a publication of the Pro… | 2021 Dec 23 | 33739538
Identification and Functional Characterization of Divergent 3’-Phosphate tRNA Ligase From Entamoeba histolytica
Ruofan Peng, Shigeo Yoshinari, ..., Tomoyoshi Nozaki
Frontiers in Cellular and Infection Microb… | 2021 Dec 17 | 34976851
Flagella, Type I Fimbriae and Curli of Uropathogenic Escherichia coli Promote the Release of Proinflammatory Cytokines in a Coculture System
Rubí Vega-Hernández, Sara A. Ochoa, ..., Azucena Mora
Microorganisms | 2021 Oct 27 | 34835359
Control of Methicillin-Resistant Staphylococcus aureus Strains Associated With a Hospital Outbreak Involving Contamination From Anesthesia Equipment Using UV-C
Sara A. Ochoa, Ariadnna Cruz-Córdova, ..., Juan Xicohtencatl-Cortes
Frontiers in Microbiology | 2020 Dec 14 | 33381094
Enzyme catalysis prior to aromatic residues: reverse engineering of a dephosphoCoA kinase
Makarov Mikhail, Meng Jingwei, ..., Hlouchová Klára
bioRxiv | 2020 Nov 11
The interplay of protein-ligand and water-mediated interactions shape affinity and selectivity in the LAO binding protein.
Renan Vergara, Sergio Romero-Romero, ..., Daniel Alejandro Fernández-Velasco
The FEBS journal | 2020 Oct 15 | 31348608
The CCAAT box in the proximal SERCA2 gene promoter regulates basal and stress-induced transcription in cardiomyocytes.
Jorge Fragoso-Medina, Gabriela Rodriguez, Angel Zarain-Herzberg
Molecular and cellular biochemistry | 2018 Jul 12 | 28884444
The first description of a hormone‐sensitive lipase from a basidiomycete: Structural insights and biochemical characterization revealed Bjerkandera adusta Ba EstB as a novel esterase
María del Rayo Sánchez‐Carbente, Ramón Alberto Batista‐García, ..., Jorge Luis Folch‐Mallol
MicrobiologyOpen | 2017 Mar 01 | 28251842
A transporter for abiotic stress and plant metabolite resistance in the ectomycorrhizal fungus Tricholoma vaccinum.
Ines Schlunk, Katrin Krause, ..., Erika Kothe
Environmental science and pollution resear… | 2016 Jul 26 | 25563836
Genotyping of Toxic Pufferfish Based on Specific PCR-RFLP Products As Determined by Liquid Chromatography/Quadrupole-Orbitrap Hybrid Mass Spectrometry
Hajime Miyaguchi, Tadashi Yamamuro, ..., Shinichi Suzuki
Journal of Agricultural and Food Chemistry | 2015 Oct 17
Modulation of ethanol stress tolerance by aldehyde dehydrogenase in the mycorrhizal fungus Tricholoma vaccinum.
Theodore Asiimwe, Katrin Krause, ..., Erika Kothe
Mycorrhiza | 2014 Aug 06 | 22159964
Human Vav1 expression in hematopoietic and cancer cell lines is regulated by c-Myb and by CpG methylation.
Lena Ilan, Shulamit Katzav
PLoS ONE | 2012 May 29 | 22253833
The crystal structure of death receptor 6 (DR6): a potential receptor of the amyloid precursor protein (APP).
Miriam Kuester, Steffen Kemmerzehl, ..., Manuel E Than
Journal of molecular biology | 2011 Jul 22 | 21463639
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