Total RNA in trophoblasts and placental chorionic tissues was extracted using a Trizol kit (Invitrogen; Thermo Fisher Scientific, Waltham, MA), and RNA concentration was tested by Nanodrop 2000 (Thermo Fisher Scientific). Takara Taq polymerase (Takara Bio, Inc., Otsu, Japan) was used for DNA amplification. Commentary DNA was synthesized using a PrimeScript RT kit (Takara Biotechnology, Co., Ltd., Dalian, China). qPCR was performed with the application of the SYBR Premix EX Taq Kit (Takara). The expression of miR‐149 was normalized to the endogenous expression of U6, and the relative expression of mRNAs was normalized to GAPDH. The relative expression of target genes was evaluated by the 2−ΔΔCt method. The PCR primers are listed in Table 2 .
Takara taq polymerase
Manufactured by Takara Bio
Sourced in Japan, China
Takara Taq polymerase is a thermostable DNA polymerase enzyme used for DNA amplification in PCR (Polymerase Chain Reaction) applications. It catalyzes the synthesis of new DNA strands complementary to a template DNA strand.
Automatically generated - may contain errors
Lab products found in correlation
Sybr premix ex taq kit, by Takara Bio (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Primescript rt kit, by Takara Bio (1 mentions)
Trizol kit, by Thermo Fisher Scientific (1 mentions)
Qiamp dna blood mini kit, by Qiagen (1 mentions)
Takara la taq dna polymerase, by Takara Bio (1 mentions)
Abi3730xl, by Sangon (1 mentions)
Qiaxcel advanced system, by Qiagen (1 mentions)
Maxwell 16 tissue dna purification kit, by Promega (1 mentions)
Pgem t vector, by Promega (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Dna ligation mighty mix, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
8 protocols using takara taq polymerase
1
Trophoblast RNA Extraction and Analysis
2
Genotyping of MODY Genetic Variants
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The remaining blood samples were stored at −20°C until extracted for genomic DNA using a QiAmp DNA Blood Mini Kit (QIAGEN, Hilden, Germany). The genotyping of rs5219 was performed as previously described.[20 (link)] Rs2285676 was genotyped using restriction fragment length polymorphism polymerase chain reaction (PCR) with BcnI enzyme (Thermo Scientific, Waltham, MA, United States). The detailed protocol for genotyping rs2285676 is described in Supplementary Table S1, http://links.lww.com/MD/H879 . Rs757110 and rs1799859 were genotyped using a tetra-primer amplification refractory mutation system PCR. The sequence of primers and their ratios in the PCR mix are listed in Supplementary Table S2, http://links.lww.com/MD/H880 . All the PCRs were performed with Takara Taq polymerase (TakaraBio, San Jose, CA, United States) in a SimpliAmp thermal cycler (Thermo Scientific) under the following conditions: initial denaturation at 98°C for 3 minutes, followed by 30 cycles of 98°C for 15 seconds (denaturation), 60°C for 20 seconds (annealing), 72°C for 40 seconds (elongation), and 72°C for 2 minutes (final elongation).
Thirty random DNA samples were chosen for direct sequencing of the genetic regions containing the 4 SNPs. The protocol for direct sequencing was described previously.[21 (link)–23 (link)] The results of sequencing were used as controls for the compatibility of PCR genotyping.
Thirty random DNA samples were chosen for direct sequencing of the genetic regions containing the 4 SNPs. The protocol for direct sequencing was described previously.[21 (link)–23 (link)] The results of sequencing were used as controls for the compatibility of PCR genotyping.
3
Amplification and Sequencing of DNA Samples
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The DNA from each of the three species was amplified using eight pairs of universal primers, as described in Zhang et al. [46 (link)], but there were still some vacancies. We then used Primer Premier 5.0 to design species-specific primers based on known sequences from universal primers [47 (link)] (Table S1 ) and used normal PCR (product length <3000 bp) as well as long PCR (product length >3000 bp) methods for amplification [46 (link)]. Takara Taq polymerase and Takara LATaq DNA polymerase were used, respectively (Takara, Dalian, China), in a 50 µL reaction volume. Reaction systems and cycling conditions for normal PCR and long PCR were as described in Zhang et al. [46 (link)]. All PCR products were sequenced in both directions using the primer-walking method and ABI3730XL by Sangon Biotech Company (Shanghai, China).
4
Microsatellite Genotyping of Thymic Lymphoma
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Genomic DNA was extracted from thymic lymphoma cells using the Maxwell 16 Tissue DNA Purification kit (Promega, Madison, WI, USA). PCR was performed using TAKARA Taq polymerase (Takara Bio Inc., Otsu, Japan) and primers for microsatellite markers (S1 Fig and S1 Table ). The PCR program consisted of 94°C for 3 min, cycles of denaturation at 94°C for 30 sec, annealing for 30 sec, and extension at 72°C for 15 sec, followed by 72°C for 5 min (see S1 Table for details). PCR products were analyzed by electrophoresis with 3% NuSieve 3:1 agarose gels (FMC, Rockland, MA, USA) or by capillary electrophoresis (QIAxcel Advanced System; Qiagen, Hilden, Germany).
5
Profiling DNA Methylation in Myeloid Cells
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Extracted DNA from sorted myeloid cells was subjected to bisulphite treatment via EZ DNA Methylation Gold Kit (Zymo Research, California, USA), followed by PCR amplification using TaKaRa Taq polymerase (TaKaRa Bio Inc., Shiga Prefecture, Japan) for PD-L1, TIM-3, galectin-9, VISTA, ARG1, MMP9 and TGF-β1. The PCR products were cloned into pGEM-T-vector (Promega Corporation, Wisconsin, USA) using DNA Ligation Mighty Mix (TaKaRa Bio). Methylation primers were designed by MethPrimer database (https://www.urogene.org/methprimer/index1.html ). Sequences are listed in Supplementary Table 1b. Seven colonies were picked from each sample and plasmid extraction was performed using GENEJET Plasmid miniprep Kit (Thermo Fisher Scientific). M13 reverse primer was used for Sanger sequencing (Supplementary Table 1c).
6
EGFR Exon 20 SNP Genotyping
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The exon 20 of EGFR gene harbouring the SNP was amplified from 364 DNA samples (180 oral cancer patients and 184 healthy controls) using the sequence specific primers EGFR Fwd:5′- M13- CCTTCTGGCCACCATGCGAA-3′ and Rev:5′-CGCATGTGAGGATCC TGGCT -3′ (where M13 in the EGFR Fwd is the universal sequencing primer 5′-tgtaaaacgacggccagt-3′). Polymerase chain reaction (PCR) was carried in 30 μL volume containing 100 ng of genomic DNA, 1X PCR buffer, 1.5 mM MgCl2, 100 μM dNTP (Takara, Japan), 80 nM primers (Sigma, India) and 0.5U of Takara Taq polymerase (Takara, Japan). The PCR is performed using the following thermal conditions: 94 °C for 2 min for initial denaturation, followed by 40 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, and 7 min of final extension at 72 °C. The PCR products (298 bp) were electrophoresed in 2% agarose gel. The purified PCR products were sequenced at Macrogen Inc, South Korea.
7
Quantitative Analysis of miR-629 Expression
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RNA was extracted from GC and paracarcinoma tissues, and SGC-7901 cells using the TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, RNA concentration was determined using Nanodrop 2000 (Thermo Fisher Scientific, Inc.). Takara Taq polymerase (Takara Bio, Inc., Otsu, Japan) was used for DNA amplification. cDNA was synthesized using the PrimeScript RT reagent kit (Takara Biotechnology, Co., Ltd., Dalian, China) at 37°C for 15 min. qPCR was performed using the SYBR Premix EX Taq™ kit (Takara Bio, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles at 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec, and then a final extension at 72°C for another 10 min. The expression of miR-629 was normalized to the endogenous expression of U6 and the relative expression of protein mRNA was normalized to GAPDH. The 2−ΔΔCq method was used to analyze mRNA expression (23 (link)). The sequences of the primers were listed in Table I .
8
Optimized Multiplex PCR Protocols
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For each of primer pair, PCRs were performed twice, each time with a different Taq enzyme and reaction buffer. All the primer pairs were amplified in the first round experiment with 20 μl reaction volumes containing 0.5 U of TaKaRa Taq polymerase (Code No.: R001A, TaKaRa, Dalian, China), 2 μl of 10 × PCR Buffer (Mg2+ plus), 0.2 μl of dNTP (2.5 mM each), 0.4 μM primer, and 50 ng of genomic DNA. Then the no bands or weak bands primers were used in the second round PCR reaction using TAKaRa LA Taq polymerase with GC buffer (Code No.: RR02AG, TaKaRa, Dalian, China) according to the manufacturer’s instructions. SSRs were amplified on Heijingang Thermal Cycler (Eastwin, Beijing, China). Under the following conditions: 5 min initial denaturation at 95°C; 35 cycles of 30 s at 95°C, 30 s at the optimized annealing temperature (Table
1 ), 45 s of elongation at 72°C, and a final extension at 72°C for 10 min. PCR products were tested for polymorphism using 6% denaturing polyacrylamide gels and visualized by silver nitrate staining.
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