Total RNA was extracted from cells or tissue samples by using the RNeasy Plus Mini kit (Qiagen, Inc.) according to the manufacturer's protocol. The 20 µl RT reactions were performed using a PrimeScript® RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) and incubated for 30 min at 37°C and 5 sec at 85°C. Subsequently, 2 µl of diluted RT product was mixed with 23 µl reaction buffer (Takara Inc.) to a final volume of 25 µl. The primer sequences are presented in Table I . All reactions labeled with SYBRGreen (Takara Inc.) were carried out using an Eppendorf Mastercycler EP Gradient S (Eppendorf) under the following conditions: 95°C for 30 sec, followed by 45 cycles of 95°C for 5 sec and 60°C for 30 sec. The expression levels of detected RNAs were normalized to GAPDH using the comparative 2−ΔΔCq method (23 (link)).
Reaction buffer
Manufactured by Takara Bio
Sourced in China, Japan
Reaction buffer is a solution designed to provide an optimal chemical environment for a specific enzymatic or biochemical reaction. It typically contains a combination of buffering agents, salts, and cofactors to maintain the desired pH, ionic strength, and other conditions necessary for the target reaction to occur efficiently.
Automatically generated - may contain errors
Lab products found in correlation
Mastercycler ep gradient s, by Eppendorf (4 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Rneasy plus mini kit, by Qiagen (3 mentions)
Hotstartaq polymerase, by Takara Bio (2 mentions)
Ez dna methylation gold kit, by Zymo Research (2 mentions)
Sybr green, by Takara Bio (1 mentions)
Gelred, by Biotium (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Prime script rt reagent kit, by Merck Group (1 mentions)
Taq dna polymerase, by Takara Bio (1 mentions)
50 bp dna ladder, by Takara Bio (1 mentions)
Primescript rtase, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Pngase f, by Takara Bio (1 mentions)
Axygen axyprep dna gel extraction kit, by Corning (1 mentions)
Q5 high fidelity dna polymerase, by Takara Bio (1 mentions)
Miseq system, by Illumina (1 mentions)
Plant rna purification reagent, by Thermo Fisher Scientific (1 mentions)
Pentr sd d topo vector, by Thermo Fisher Scientific (1 mentions)
Primestar hs dna polymerase, by Takara Bio (1 mentions)
15 protocols using reaction buffer
1
RNA Extraction and qRT-PCR Analysis
2
Quantifying Gene Expression in Breast Cancer
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Total RNA was extracted from breast cancer tissues or cells by using the RNeasy plus mini kit (Qiagen) according to the manufacturer’s protocol. RT and qPCR kits were used to evaluate the expression of target RNAs. RT (20 μl) reactions were performed using the PrimeScript® RT reagent kit (Takara, Dalian, China) and incubated for 30 min at 37 °C followed by 5 s at 85 °C. For qPCR, 2 μl of diluted RT product was mixed with 23 μl reaction buffer provided by Takara (Takara Inc., Dalian, China) to a final volume of 25 μl. All reactions were carried out using an Eppendorf Mastercycler EP Gradient S (Eppendorf, Germany) under the following conditions: 95 °C for 30 s followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s. The internal expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for the normalization of detected RNAs using the comparative 2-ΔΔCq method. The primer sequences for qPCR are presented in Additional file 1 : Table S1.
3
Intracellular Nuclease Activity Assay
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To assess intracellular nuclease activity, crude enzyme extracts of WP3NR strains were incubated together with DNA substrate. In particular, the WP3NR strains from cultures at the mid-logarithmic phase (OD600 = 1.0) were pelleted by centrifugation. The harvested cell pellets were resuspended in lysis buffer [500 mM NaCl, 10% glycerol (w/v), 20 mM Tris–HCl (pH 8.0)] and sonicated on ice. The cell lysates were subsequently centrifuged at 10,000 × g for 20 min at 4 °C, after which the supernatants were obtained. Nine microliters of the crude enzyme extracts were incubated together with approximately 100 ng of purified DNA (PCR-amplified 16 S rRNA gene and genomic DNA of S. piezotolerans WP3) in reaction buffer (Takara, Dalian, China). The reaction mixtures were incubated at 20 °C for 1 h and then separated on a 1% agarose gel. The DNA was then stained with GelRed (Biotium, Hayward, USA) and visualized by a gel imaging system (Tanon, Shanghai, China).
4
Quantitative PCR Analysis of Target RNAs
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Total RNA was extracted from BCa tissues or cells using the Qiagen RNeasy Mini kit according to the manufacturer's protocol (Qiagen GbmH). RT and qPCR kits were used to evaluate the expression of target RNAs. RT reactions (20 µl) were performed using the PrimeScript® RT reagent kit (Takara Biotechnology Co., Ltd.) and incubated for 30 min at 37°C followed by 5 sec at 85°C. For qPCR, 2 µl diluted RT product was mixed with 23 µl reaction buffer (Takara Biotechnology Co., Ltd.) to a final volume of 25 µl. All reactions were performed using an Eppendorf Mastercycler EP Gradient S (Eppendorf) under the following conditions: Initial denaturation at 95°C for 30 sec; and 45 cycles of denaturation (95°C for 5 sec), and annealing and elongation (60°C for 30 sec). The transcript expression of GAPDH was used for the normalization of detected RNAs using the comparative 2-ΔΔCq method (18 (link)). The primer sequences for qPCR are presented in Table IB .
5
Quantifying Breast Cancer RNA Expression
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Total RNA of breast cancer cells or tissues were harvested through RNeasy Plus Mini Kit from Qiagen base on the manufacturer's instructions. The target RNA expression was evaluated using the kits for reverse transcription (RT) and real-time PCR. Reactions for RT were performed in a volume of 20 μL through the PrimeScript RT reagent kit (Sigma), kept at 37°C for 0.5 hours and then at 85°C for 5 s. To carry out real-time PCR, RT product was diluted with reaction buffer (Takara Inc, Dalian, China), and each reaction was performed on an EP Gradient S Eppendorf Mastercycler from Eppendorf (Germany) under the following conditions: 95 ℃ for 30 s; 45 cycles at 95 ℃ for 5 s; and 60 ℃ for 30 s. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as the internal control to normalize expression as per the method of comparative 2 -ΔΔCq . Primers are listed as follows: Mcl-1, Forward: 5′-CCAAGAAAGCTGCATCGAACCAT-3′, Reverse: 5′-CAGCACATTCCTGATGCCACCT-3′; Bim, Forward: 5'-GGCCCCTACCTCCCTACA-3', Reverse: 5'-GGGGTTTGTGTTGATTTGTCA-3'; GAPDH, Forward: 5'-AGCCACATCGCTCAGACAC-3', Reverse: 5'-GCCCAATACGACCAAATCC-3'.
6
Microsatellite Marker Amplification Protocol
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Total DNA was extracted from the silica gel-dried leaves using a modified 2X CTAB procedure (Doyle and Doyle, 1987) . The 14 SSR markers (Table 2) used in the present study were selected based on their ability to amplify DNA in this species, and the reproducibility of their products. DNA amplifications were performed in 20-mL reaction volumes containing 1X reaction buffer (TaKaRa), 10-15 ng genomic DNA, 250 mM each dNTP, 20 mM each primer, and 1 U Taq DNA polymerase (Takara). The PCR was programmed according to the following profile: 94°C for 5 min, followed by 30 cycles at 94°C for 45 s, annealing temperature for 45 s, 72°C for 45 s, and a final extension at 72°C for 5 min. A 1-µL aliquot of each sample was separated on 8% denaturing polyacrylamide gel using silver staining. A 50-bp DNA ladder (Takara) was used to identify alleles (Figure 1).
7
Quantitative PCR Analysis of Cardiac Genes
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One microgram of total RNA, isolated from rat ventricular tissue or cultured cells using TRIzol (Invitrogen), was reversely transcribed into cDNA using a 5×PrimeScript RT master mix, including PrimeScript RTase, RNase inhibitor, random 6-mers, oligo dT (deoxythymine) primer, dNTP (deoxy-ribonucleoside triphosphate) mixture, and reaction buffer (Takara, Japan). 49 (link) Expressions of β-MHC and ANP genes were quantified by real-time quantitative PCR (ABI 7500 Fast) using the primers listed in Table S3.
8
Targeted CpG Methylation Analysis via NGS
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DNA methylation level was analysis by MethylTarget™, an NGS-based multiple targeted CpG methylation analysis method. Specifically, the genomic regions of interest were analyzed and transformed to bisulfite-converted sequences by geneCpG software. PCR primer (Table 1 ) sets were designed with the Methylation Primer software from bisulfate converted DNA [7 (link)].
Genomic DNA (400 ng) was subjected to sodium bisulfite treatment using EZ DNA Methylation™-GOLD Kit (Zymo Research) according to manufacturer's protocols. Multiplex PCR was performed with optimized primer sets combination. A 20 μl PCR reaction mixture was prepared for each reaction and included 1x reaction buffer (Takara), 3 mM Mg2+, 0.2 mMdNTP, 0.1 μM of each primer, 1 U HotStarTaqpolymerase (Takara), and 2 μl templates DNA. The cycling program was 95°C for 2 min; 11 cycles of 94°C for 20 s, 63°C for 40 s with a decreasing temperature step of 0.5°C per cycle, and 72°C for 1 min and then followed by 24 cycles of 94°C for 20 s, 65°C for 30 s, and 72°C for 1 min; 72°C for 2 min [7 (link)].
Genomic DNA (400 ng) was subjected to sodium bisulfite treatment using EZ DNA Methylation™-GOLD Kit (Zymo Research) according to manufacturer's protocols. Multiplex PCR was performed with optimized primer sets combination. A 20 μl PCR reaction mixture was prepared for each reaction and included 1x reaction buffer (Takara), 3 mM Mg2+, 0.2 mMdNTP, 0.1 μM of each primer, 1 U HotStarTaqpolymerase (Takara), and 2 μl templates DNA. The cycling program was 95°C for 2 min; 11 cycles of 94°C for 20 s, 63°C for 40 s with a decreasing temperature step of 0.5°C per cycle, and 72°C for 1 min and then followed by 24 cycles of 94°C for 20 s, 65°C for 30 s, and 72°C for 1 min; 72°C for 2 min [7 (link)].
9
Targeted DNA Methylation Analysis by NGS
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DNA methylation level was analysis by MethylTargetTM (Genesky Biotechnologies Inc., Shanghai, China), an NGS-based multiple Targeted CpG methylation analysis method. Specifically, the genomic regions of interest were analyzed and transformed into bisulfite-converted sequences by geneCpG software. PCR primer sets were designed with the Methylation Primer software from bisulfate converted DNA.
Genomic DNA (400 ng) was subjected to sodium bisulfite treatment using EZ DNA Methylation™-GOLD Kit (Zymo Research) according to the manufacturer's protocols. Multiplex PCR was performed with optimized primer sets combination. A 20 μl PCR reaction mixture was prepared for each reaction and included 1x reaction buffer (Takara), 3 mM Mg2+, 0.2 mM dNTP, 0.1 μM of each primer, 1U HotStarTaq polymerase (Takara) and 2 μl template DNA. The cycling program was 95°C for 2 min; 11 cycles of 94°C for 20 s, 63°C for 40s with a decreasing temperature step of 0.5°C per cycle, 72°C for 1 min; then followed by 24 cycles of 94°C for 20 s, 65°C for 30 s, 72°C for 1 min; 72°C for 2 min.
Genomic DNA (400 ng) was subjected to sodium bisulfite treatment using EZ DNA Methylation™-GOLD Kit (Zymo Research) according to the manufacturer's protocols. Multiplex PCR was performed with optimized primer sets combination. A 20 μl PCR reaction mixture was prepared for each reaction and included 1x reaction buffer (Takara), 3 mM Mg2+, 0.2 mM dNTP, 0.1 μM of each primer, 1U HotStarTaq polymerase (Takara) and 2 μl template DNA. The cycling program was 95°C for 2 min; 11 cycles of 94°C for 20 s, 63°C for 40s with a decreasing temperature step of 0.5°C per cycle, 72°C for 1 min; then followed by 24 cycles of 94°C for 20 s, 65°C for 30 s, 72°C for 1 min; 72°C for 2 min.
10
Deglycosylation of DENV-2 Particles
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To deglycosylate the purified DENV-2 particles that were produced by insect cells, 100 μg of DENV-2 particles were denatured by heating at 100°C for 10 min in the presence of 0.5% SDS and 40 mM dithiothreitol. After cooling, 1 μl of 500,000 U/ml PNGase F (Takara, Japan) was added, and the mixture was incubated in 5× reaction buffer (Takara, Japan) at 37°C for 1 h. Subsequently, the samples were subjected to SDS-PAGE and western blot analyses using anti-DENV hyperimmune mouse serum as described elsewhere.
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