PCR amplifications were performed on a PC320 (Astec, Japan) or GeneAtlasG02 (Astec) thermocycler. Amplification of PCR products from DNA derived from leaves and dried fruits was achieved using a final reaction volume of 20 μL containing 0.5 μL of genomic DNA template, 2.0 μL of 10× PCR Buffer for KOD Dash, 0.2 mM of dNTPs, 0.2 μM each of forward and reverse primers (A1/A2, C1/C2), and 0.5 U of KOD Dash® (Toyobo, Japan). The temperature cycling program for PCR consisted of 1 min at 94°C, followed by 30 cycles of 30 s at 94°C, 2 s at annealing temperature and 30 s at 74°C, with a final extension for 1 min at 72°C. To amplify PCR products from DNA derived from resin-containing portions of agarwood, the final reaction volume of 20 μL contained 2 μL of genomic DNA template, 2.0 μL of 10× PCR Buffer for KOD -Plus-, 0.2 mM of dNTPs, 1.2 μL of MgSO4, 0.3 μM each of forward and reverse primers (A1/A2, C1/C2), and 0.4 U of KOD -Plus-(Toyobo). The temperature cycling program for PCR consisted of 2 min at 94°C, followed by 35 cycles of 10 s at 98°C, 30 s at annealing temperature and 30 s at 68°C. The annealing temperatures for PCR depended on the combinations of primers (Table 4).
Kod dash
Manufactured by Toyobo
Sourced in Japan
The KOD Dash is a high-performance DNA polymerase designed for rapid and accurate DNA amplification. It is suitable for a wide range of PCR applications, including routine, long-range, and high-fidelity PCR.
Automatically generated - may contain errors
Lab products found in correlation
Kod plus, by Toyobo (1 mentions)
Pc 320, by Astec (1 mentions)
Geneatlas g02, by Astec (1 mentions)
Ex taq, by Takara Bio (1 mentions)
Fas 4 gel imaging system, by Nippon Genetics (1 mentions)
Human multiple tissue cdna panels 1 and 2, by Takara Bio (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Stepone system, by Thermo Fisher Scientific (1 mentions)
Oligo dt primer, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Dig digoxigenin rna labeling kit, by Merck Group (1 mentions)
Anti dig antibody conjugated with alkaline phosphatase, by Merck Group (1 mentions)
Sp6 rna polymerase, by New England Biolabs (1 mentions)
Gdna eraser, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Trizol, by Merck Group (1 mentions)
Ptagbfp n, by Evrogen (1 mentions)
12xcsl d1egfp, by Addgene (1 mentions)
Poly a tailing kit, by Thermo Fisher Scientific (1 mentions)
Mmessage mmachine t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
8 protocols using kod dash
1
PCR Optimization for Agarwood Samples
2
PCR Assay Template Generation
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Templates used in the PCR assay were generated by inserting tdTomato (Clontech) between the KpnI and XbaI cutting sites, or RNR1 between the KpnI and XhoI cutting sites of the plasmid pUC119. Template DNA concentrations were optimized to give signals of nearly equivalent apparent strength under ethidium bromide (EtBr) staining. All PCR reactions were performed with ScaI-linearized pUC119 templates for 12 cycles. The set of primers flanking the multi-cloning site of plasmid pUC119 used for all PCR reactions was: pUC119-MCS-Fwd, 5′-TTGTGTGGAATTGTGAGCGG-3′; pUC119-MCS-Rev, 5′-TGCAAGGCGATTAAGTTGGG-3′. KOD Dash (TOYOBO) or Ex Taq (Takara Bio.) polymerases were used for PCR. Gels were photographed with a FAS-IV gel imaging system (Nippon Genetics) and band intensities were measured with ImageJ software. All band intensities were normalized against the control band with the weakest EtBr signal (left side of gel; amplified with 200 µM dNTPs). Relative DNA levels (EtBr signal %) at each dNTP concentration were calculated as Anorm/Anorm + Bnorm.
3
Quantifying CD73 Expression in Human Tissues
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Total RNAs were extracted from cells using the RNeasyMini Kit (Qiagen, Hilden, Germany). cDNA was synthesized using Superscript III and an oligo(dT) primer (Life Technologies Corp.). Human Multiple Tissue cDNA Panels I and II, and the Human Fetal Multiple Tissue cDNA Panel (Clontech; Mountainview, CA) were used as normal tissue cDNAs. PCR was performed using KOD Dash (TOYOBO, Osaka, Japan) to detect CD73. The primer sequences used were 5′- TGATGAACGCAACAATGGAATC-3′ and 5′- ATGGCAGTGACTTCCTGTGG-3′. The PCR mixture was denatured at 94°C for 2 min, followed by 30 cycles at 94°C for 15 sec, at 58°C for 2 sec, and at 74°C for 30 sec. GAPDH was used as an internal control. Real-time PCR was performed using the StepOne system (Life Technologies Corp.). Primers and probes were designed using the TapMan Gene expression assay (Life Technologies Corp.). Thermal cycling was performed with 40 cycles of 95°C for 1 sec, followed by 60°C for 20 min. Each experiment was done in triplicate and normalized to the GAPDH gene as an internal control.
4
Whole-Mount In Situ Hybridization for Zebrafish
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Whole-mount in situ hybridization was performed as described previously [34 (link)]. The liver-type fatty acid-binding protein 10a (fabp10a) and hepatocyte nuclear factor 3-gamma (foxa3) were amplified by PCR reaction with KOD Dash (TOYOBO) with the primer sets listed in Table S1 . This was followed by subcloning to the pTAC-2 TA vector (BioDynamics) and in vitro transcription with SP6 RNA polymerase (New England Biolabs, Ipswich, MA, USA) with the DIG (digoxigenin) RNA Labeling Kit (Sigma-Aldrich) to obtain antisense RNA probes. Four percent paraformaldehyde (PFA)-fixed embryos were hybridized with digoxigenin-incorporated RNA probes at 65 °C overnight. Following hybridization and washing, the embryos were incubated with an anti-DIG antibody conjugated with alkaline phosphatase (Sigma-Aldrich) at 4 °C overnight. Color reaction was performed via incubation in NBT/BCIP Ready-to-Use Tablets substrate (Sigma-Aldrich).
5
Quantitative PCR Analysis of GLUT4 and LDLR
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The effects of BBR on mRNA level of glucose transporter 4 (GLUT4) and low density lipoprotein receptor (LDLR) were determined by semiquantitation polymerase chain reaction (RT-PCR) in hamsters. RT-PCR was performed as previously described [12 (link), 13 (link)]. Total RNA was isolated from the liver and skeletal muscle using TRIzol (Sigma), and cDNA was synthesized with the PrimeScript RT Reagent Kit and gDNA Eraser (Perfect Real Time; Takara, Shiga, Japan). KOD Dash (Toyobo, Osaka, Japan) was used for RT-PCR amplification. The value of gene expression was calculated after normalization to β-actin. The primers used in the experiment are listed in the Supplemental Material (available online at http://dx.doi.org/10.1155/2015/313808 ).
6
Identification and Characterization of Biomolecules
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Purified CRP was purchased from OriGene Technologies Inc. (Rockville, Maryland, United States), and purified LDH-5 was purchased from Meridian Life Science Inc. (Tennessee, United States). Purified LDH-1 isoenzyme was purchased from RayBiotech Life, United States. Magnetic beads for the immobilization of the target and recovery of biotinylated DNA—namely, Dynabeads MyOne Carboxylic Acid magnetic beads, and Dynabeads MyOne SA C1 magnetic beads—were purchased from Invitrogen (Carlsbad, CA, United States). KOD Dash (TOYOBO, Japan) was used for PCR and incorporating modified bases. Synthetic compounds used as primers, random pools, and aptamer clone templates were purchased from Integrated DNA Technologies MBL KK (IDT-MBL KK, Japan). Research grade materials were used for other reagents. The dUadTP, dUguTP, and dAadTP were synthesized using previously reported methods [17 (link),52 (link),53 (link)].
7
Amplification and Transcription of EGFP and BFP mRNAs
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Template DNAs for EGFP and BFP mRNAs were prepared via PCR using KOD Dash (TOYOBO, Japan) with 12XCSL-d1EGFP (Addgene, Watertown, MA, USA) and pTagBFP-N (Evrogen, Russia) vectors as templates. The template DNAs for EGFP and BFP mRNAs were amplified using the following primers (sequence of the T7 promoter is underlined): 5′-CCG GGT AAT ACG ACT CAC TAT AG G GAC ACA ACT GTG TTT ACT TGC-3′ and 5′-GAT GCT ATT GCT TTA TTT GTA AC-3′ for EGFP, and 5′-CCG GGT AAT ACG ACT CAC TAT AG G TCT ATA TAA GCA GAG CTG G-3′ and 5′-GTT AAC AAC AAC AAT TGC ATT C-3′ for BFP. Capped mRNAs were prepared via in vitro transcription using the mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen) and a polyA addition with the Poly (A) Tailing Kit (Invitrogen) according to the manufacturer's protocols.
8
Detection of Avian Viruses in Poultry
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PCR tests were performed separately using pooled intestinal tract and oviduct samples to detect AAV; pooled parenchyma and respiratory organs for infectious bronchitis virus (IBV ); pooled parenchyma and bone marrow for chicken anemia virus (CAV ); and pooled intestinal tract and oviduct for EDSV. Samples were manually homogenized using a sterile mortar and pestle to a concentration of 30% with Hanks' solution (Nissui Pharma, Tokyo, Japan) containing Penicillin-G (Meiji Seika Pharma Co., Ltd., Tokyo, Japan) at 400 U/mL and streptomycin (Meiji Seika Pharma Co., Tokyo, Japan) at 0.4 mg/mL. The mixture was centrifuged at 3,000 × g for 10 min. DNA was extracted using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions. PCR was performed using KOD Dash (Toyobo Co. Ltd., Osaka, Japan) following the manufacturer's instructions and using previously described primers and conditions with slight modifications: for AAV, the method followed by Mase et al., 2009 (link); for CAV, Yilmaz et al., 2001 (link); for EDSV, Begum et al., 2013 ; and for IBV, Mase et al., 2004 (link) (Supplementary Table 1 ).
In the case of sample-inoculated chick kidney cells (CKC ), the supernatant was used for DNA extraction. AAV-PCR was performed using KOD Dash as previously described (Mase et al., 2009 (link)).
In the case of sample-inoculated chick kidney cells (
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