The RNA used for qRT-PCR was from the same set RNA for northern blotting analysis. The RNA was then reverse transcribed into cDNA using the PrimeScript RT reagent Kit (Takara, Japan). The qRT-PCR was performed in a 15 μl reaction mixture consisting of 1.5 μl 1 × SYBR Green (Invitrogen, United States), 1.5 μl PCR buffer, 0.3 μl 10 mM dNTPs (Takara, Japan), 0.3 μl Taq, 0.3 μl ROX DYE2 (Vazyme, China), 1.5 μl 2 mM each primer and 2 μl appropriate diluted cDNA. The conditions for qRT-PCR were as follow: 94°C for 3 min, then 40 cycles at 94°C for 30 s and 58°C for 30 s, followed by 72°C for 35 s for PCR amplification. Transcript levels of each gene were measured by the Applied Biosystems 7500 (Applied Biosystems, United States) according to the manufacturer’s instructions. 18S rRNA was used as a quantitative control in the qRT-PCR analysis. Primers used in this study are listed in Supplementary Table S4 .
Dntps
Manufactured by Takara Bio
Sourced in Japan, China, United States
DNTPs are a set of four nucleotides (dATP, dCTP, dGTP, and dTTP) that serve as the building blocks for DNA synthesis. They are essential components used in various molecular biology techniques, such as DNA amplification, sequencing, and labeling.
Automatically generated - may contain errors
Lab products found in correlation
Restriction enzyme, by Takara Bio (17 mentions)
Taq dna polymerase, by Takara Bio (15 mentions)
Taq polymerase, by Takara Bio (12 mentions)
Dna polymerase, by Takara Bio (12 mentions)
T4 ligase, by Takara Bio (10 mentions)
T4 dna ligase, by Takara Bio (9 mentions)
Mgcl2, by Takara Bio (9 mentions)
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Rnase inhibitor, by Takara Bio (7 mentions)
Betaine, by Merck Group (6 mentions)
Ex taq dna polymerase, by Takara Bio (6 mentions)
Ex taq polymerase, by Takara Bio (6 mentions)
Dna marker, by Takara Bio (6 mentions)
Bst dna polymerase large fragment, by New England Biolabs (6 mentions)
M mlv reverse transcriptase, by Takara Bio (5 mentions)
Reverse transcriptase, by Takara Bio (5 mentions)
Thermopol reaction buffer, by New England Biolabs (5 mentions)
Bst dna polymerase, by New England Biolabs (5 mentions)
Pmd18 t, by Takara Bio (4 mentions)
M mlv reverse transcriptase, by Promega (4 mentions)
149 protocols using dntps
1
Quantitative Real-Time PCR Protocol
2
Mitochondrial COI Gene Amplification from Spiny Lobster
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was extracted from the muscle of the pereiopod of all 17 spiny lobsters. DNA was extracted using the AccuPrep® Genomic DNA Extraction Kit (Bioneer, Daejeon, South Korea), following the manufacturer’s instructions. Concentration and purity of the extracted DNA were measured using a Thermo Scientific™ NanoDrop™ One microvolume UV-Vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
A polymerase chain reaction (PCR) was performed to amplify the mitochondrial COI gene region using the HCO1490/LCO2198 universal primers, specially designed for invertebrates (Folmer et al., 1994 (link)). PCR was performed using a 50 µL reaction mixture, consisting of 100 ng genomic DNA, 0.25 µL Taq polymerase (Takara Bio Inc., Shiga, Japan), 5 µL 10X Ex. Taq DNA polymerase buffer (Takara Bio Inc.), 1 µL each of 10 µM forward and reverse primers, and 4 µL (2.5 mM) dNTPs (Takara Bio Inc.). The PCR thermal profile comprised an initial step of 5 min at 94 °C, followed by 30 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 45 s, followed by a final extension at 72 °C for 5 min. The amplified PCR products were separated using 1% agarose gel electrophoresis, and target bands were purified using the AccuPrep® PCR Purification Kit (Bioneer), according to the manufacturer’s instructions.
A polymerase chain reaction (PCR) was performed to amplify the mitochondrial COI gene region using the HCO1490/LCO2198 universal primers, specially designed for invertebrates (Folmer et al., 1994 (link)). PCR was performed using a 50 µL reaction mixture, consisting of 100 ng genomic DNA, 0.25 µL Taq polymerase (Takara Bio Inc., Shiga, Japan), 5 µL 10X Ex. Taq DNA polymerase buffer (Takara Bio Inc.), 1 µL each of 10 µM forward and reverse primers, and 4 µL (2.5 mM) dNTPs (Takara Bio Inc.). The PCR thermal profile comprised an initial step of 5 min at 94 °C, followed by 30 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 45 s, followed by a final extension at 72 °C for 5 min. The amplified PCR products were separated using 1% agarose gel electrophoresis, and target bands were purified using the AccuPrep® PCR Purification Kit (Bioneer), according to the manufacturer’s instructions.
3
Transcript Analysis of Developing Soybean Seeds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Parental lines FUKU and MUT were grown in a greenhouse (February to June 2018) under a long-day photoperiod (14.5 h/9.5 h) until the end of March. Developing seed was collected randomly from several plants at 40, 55, and 70 days after flowering (DAF). Total RNA was isolated from developing seed (50–100 mg) with a Total RNA Extraction Kit Mini Plant (SciTrove, Tokyo, Japan) with additional rDNase I (Takara Bio Inc., Shiga, Japan) treatment. cDNA was synthesized from 1 μg total RNA with ReverTra Ace (Toyobo, Osaka, Japan). A 5-μL aliquot (approx. 25 ng) of cDNA was used as a template. Quantitative real-time PCR was conducted as follows in a LightCycler 96 system: 95°C for 5 min for activation of enzyme, 95°C for 15 s, 60°C for 15 s, 72°C for 20 s, for a total of 40 amplification cycles. EvaGreen Dye (20× in water; Biotium, Inc., Fremont, CA, USA) was used as the fluorescent dye, and dNTPs (Takara), homemade recombinant Taq polymerase, the PCR buffer mentioned in a previous study (Yamagata et al. 2018 (link)), and 1 pmol of each gene-specific primer were used to evaluate transcript levels of target genes. Expression relative to GmACT2/7 (Glyma.19G147900), the internal reference gene, was calculated by using the 2-ΔΔCt method (Schmittgen and Livak 2008 (link)). Data from four technical replicates were analyzed. The primers used are listed in Supplemental Table 1 .
4
Rapid and Sensitive LAMP Detection of P. aroidearum
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LAMP reaction mixture contained 1.6 µM each of FIP and BIP, 0.4 µM each of LF and LB, 0.2 µM each of F3 and B3, 1.4 mM each of dNTPs (TaKaRa, Dalian, China), 1 M betaine (Sigma, St. Louis, MO, USA), 1× isothermal amplification buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0.1% Tween-20, pH 8.8), 6 mM MgSO4, 8 U of Bst 2.0 WarmStart polymerase (New England Biolabs, Beverly, MA, USA), 1 µL of template DNA, and sterile deionized water to a final volume of 25 μL. For the negative control, sterilized deionized water was used to replace the DNA template. These reaction mixtures were respectively put into 200 μL centrifuge tubes, which were placed in a T100 Thermal Cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) at a constant temperature of 65 °C for 40 min. Then, the Bst 2.0 WarmStart polymerase was heat inactivated at 80 °C for 5 min to stop the reactions. When the reaction products were cooled, 1 µL of 1/10 diluted original SYBR Green I (Solarbio, Beijing, China) was added into every tube for color reaction.
To verify whether the LAMP product was the correct target fragment, conventional PCR amplification was performed with the outer primers F3 and B3. The 218-bp fragment of P. aroidearum was amplified, and was cloned into pMD18-T (Takara, Kyoto, Japan) and sequenced.
To verify whether the LAMP product was the correct target fragment, conventional PCR amplification was performed with the outer primers F3 and B3. The 218-bp fragment of P. aroidearum was amplified, and was cloned into pMD18-T (Takara, Kyoto, Japan) and sequenced.
5
Molecular Cloning Using Restriction Enzymes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Restriction enzymes, DNA polymerase, dNTPs and all antibiotics were purchased from TaKaRa Biotech. PCR primers were synthesized by Sangon Biotech Company. All other reagents were purchased from Sigma unless specified.
6
Bacterial DNA Extraction and PCR Amplification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was extracted from the bacterial pellet using a QIAamp DNA Minikit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The PCR reaction mixture included 5 μL of 10 × PCR buffer (Perkin Elmer, Waltham, MA, USA), 3 μL of MgCl2 (1.5 mM; Perkin Elmer), 2.5 μL of dNTPs (2.5 mM; TakaraBio, Shiga, Japan), 5 μL of each primer (10 pmole/μL), 0.4 μL of Ampli Taq Gold (5 U/μL; Perkin Elmer), and 2 μL of extracted DNA in a final volume of 50 μL. The primers used in this study are listed in Table 4 . The thermal cycling conditions for PCR amplification consisted of an initial denaturation at 94 °C for 10 min, followed by 36 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 50 s, with a final elongation at 72 °C for 5 min. The amplified PCR products were electrophoresed on a 2% agarose gel and visualized using the Gel Doc system (Bio-Rad, Hercules, CA, USA).
7
Quantitative RT-PCR for DNAJC6 Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TRIzol reagent (Cat #15596018, Thermo Fisher, Waltham, MA, USA) was used to collect cells, and an RNA pellet was centrifuged. The pellet was completely dissolved in RNase-free water and quantified using a Nano-Drop spectrophotometer. RNA (1 μg), dNTPs (Cat #4030, Takara Bio, Shiga, Japan), and a random primer (Invitrogen, San Diego, CA, USA) were mixed and heated at 65 °C for 5 min. A master mix was prepared with Superscript III Reverse Transcriptase, 5× First Strand buffer, and 0.1 M DTT (Cat #18080093, Invitrogen, Carlsbad, CA, USA), and the RNA mix and master mix were combined to prepare cDNA. PCR PreMix (Cat #K2036, Bioneer, Daejeon, Korea) was mixed with 19 µL of primer (Zenotech, Daejeon, Korea) and 1 µL of cDNA. The DNAJC6 primer (forward: GTG TAC GGT GGG AGG TCT AT; reverse: CCG CCT TTC ACC ATG TCA AA) was heated at 94 °C for 4 min, followed by 30 cycles of 30 s at 94 °C, 30 s at 61 °C, and 40 s at 72 °C. A total of 20 µL of the prepared sample was loaded on an agarose gel and electrophoresed at 100 V for 30 min. The gel was analyzed using a Chemi Doc Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).
8
Nucleotide and Ligand Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
dNTPs were purchased from Takara Bio (Shiga, Japan). Ligands were dissolved in Milli-Q water or dimethyl sulfoxide. Other reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan) and used without further purification.
9
Recombinant Protein Production Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The steroids used in this study were obtained from TCI (Tokyo Chemical Industry Co., Ltd, Japan). Isopropyl-1-thio-β-D-galactopyranoside (IPTG), 1,4-dithiothreitol (DTT), and kanamycin were purchased from Duchefa Bohemie (Korea). Ampicillin, δ-aminolevulinic acid (ALA), NADH, and formate dehydrogenase were purchased from Sigma-Aldrich (Korea). All of the restriction enzymes, DNA polymerase, T4 DNA ligase, and dNTPs were purchased from Takara Bio (Japan). All other high-grade chemical products were obtained from commercially available sources.
10
Nested PCR for Canine Parvovirus Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The primers for NPCR for the detection of CPV were as per Mizak and Rzezutka [10 ]. The NPCR reaction mixture was prepared by adding 5 μL of the PCR product (from above), 2.5 μL of 10× PCR buffer (with 15 mM MgCl2), 1.0 μL each of forward and reverse primer (20 pm/μL), 1.0 μL of dNTPs (10 mM each) (Takara Bio), 0.2 μL Taq DNA polymerase (5 units/μL) Qiagen), and the final volume was made up to 25 μL by adding nuclease-free water.
Both PCR and NPCR were set at thermocycling parameters with 35 cycles of denaturation at 94°C for 60 s, annealing at 55°C for 60 s, elongation at 72°C for 150 s, and a final elongation at 72°C for 10 min. Both PCR and NPCR products (10 μL each) were subjected to agarose gel electrophoresis using 1% agarose at the rate of 5 volts/cm with Gene Ruler ladder plus 100 bp (New England Biolabs, USA). The products on gel were visualized and documented using gel documentation system (Syngene, USA).
Both PCR and NPCR were set at thermocycling parameters with 35 cycles of denaturation at 94°C for 60 s, annealing at 55°C for 60 s, elongation at 72°C for 150 s, and a final elongation at 72°C for 10 min. Both PCR and NPCR products (10 μL each) were subjected to agarose gel electrophoresis using 1% agarose at the rate of 5 volts/cm with Gene Ruler ladder plus 100 bp (New England Biolabs, USA). The products on gel were visualized and documented using gel documentation system (Syngene, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!