The full-length sequences of pre-microRNAs were amplified from genomic DNA using high-fidelity KOD-Plus-Ver.2 DNA Polymerase (Toyobo, Osaka, Japan). The amplification products were inserted into pSAK277 vectors. The target sequences of microRNAs with sizes of 200–300 bp were amplified from first-strand cDNA. The amplification fragments were then inserted into a pGreenII Dual-Luciferase microRNA Target Expression Vector18 (link). The subsequent process for introduction of the constructed vectors into Agrobacterium tumefaciens, suspension, infiltration, and culture of tobacco plants were identical to those in a previous study18 (link). Luminescence assays were conducted using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI). Firefly luciferase (Luc) and Renilla luciferase (Ren) activity levels were measured using a Cytation 3 Cell Imaging Multi-Mode Reader (BioTek, Santa Barbara, CA) for at least ten biological replicates for each assay. All primer sequences are listed in Table S14 .
Kod plus ver 2 dna polymerase
Manufactured by Toyobo
Sourced in Japan
KOD-Plus-ver.2 DNA polymerase is a high-fidelity DNA polymerase designed for accurate DNA amplification in PCR applications. It exhibits enhanced processivity and thermal stability compared to the original KOD DNA polymerase.
Automatically generated - may contain errors
Lab products found in correlation
Cytation 3 cell imaging multi mode reader, by Agilent Technologies (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Rnaiso plus reagent, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
Wizard sv gel and pcr clean up system, by Promega (1 mentions)
Takara ex taq polymerase, by Takara Bio (1 mentions)
Neb buffer 2, by New England Biolabs (1 mentions)
T7 endonuclease 1, by New England Biolabs (1 mentions)
Amino acids, by Fujifilm (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Pyruvic acid, by Fujifilm (1 mentions)
Rlm race kit, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Restriction endonuclease, by Takara Bio (1 mentions)
Kod fx dna polymerase, by Toyobo (1 mentions)
Universal dna purification kit, by Tiangen Biotech (1 mentions)
Pegfp c1, by Takara Bio (1 mentions)
10 protocols using kod plus ver 2 dna polymerase
1
Cloning and Validation of miRNA Targets
2
RNA Extraction and cDNA Synthesis
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Forty-eight hours after transfection, total RNAs were extracted
with RNAiso Plus reagent (TaKaRa, Beijing, China). cDNAs were synthesized
using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa) following
the standard protocol. KOD-Plus-Ver.2 DNA polymerase (Toyobo) was
used to amplify the corresponding cDNA sequences flanking the joint
point of dsDNAs.
with RNAiso Plus reagent (TaKaRa, Beijing, China). cDNAs were synthesized
using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa) following
the standard protocol. KOD-Plus-Ver.2 DNA polymerase (Toyobo) was
used to amplify the corresponding cDNA sequences flanking the joint
point of dsDNAs.
3
Genetic Sequencing of MYH7 and HSPA6 Genes
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Genomic DNA was extracted from whole peripheral blood leukocytes. The MYH7 and HSPA6 genes were amplified by using TaKaRa Ex Taq polymerase (Takara Bio, Shiga, Japan) and KOD Plus Ver. 2 DNA polymerase (TOYOBO, Osaka, Japan), respectively, according to the corresponding manufacturers’ instructions. PCR products were direct-sequenced on the 3730xl DNA analyzer (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA) after purification by Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). Primer sequences for PCR and sequencing of the MYH7 and HSPA6 genes are available on request.
4
Production and Manipulation of PhaB Proteins
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DNA manipulations were carried out according to standard procedures, and PCR reactions were performed with KOD-Plus ver.2 DNA polymerase (Toyobo, Osaka). The sequences of oligonucleotide primers used in this study are shown in Additional file 1 : Table S4.
pColdII-phaB1, pColdII-phaB2, and pET15b-phaB3 vectors for overproduction of N-His6-tagged PhaB1, PhaB2, and PhaB3 in E. coli, respectively, were constructed as described in Supporting Information. Site-directed mutagenesis of phaB1 was carried out by QuickChange protocol. A DNA fragment consisting of a tandem of had and crt2 was prepared by fusion PCR. Several plasmids were constructed by blunt-end ligation of a DNA fragment with a linear fragment of the backbone plasmid prepared by inverse PCR. pBPP-ccrMeJAc-emd was constructed by replacement of phaJ4a in pBPP-ccrMeJ4a-emd [23 (link), 44 (link)] by phaJAc amplified with pEE32 [10 (link)] as a template. Plasmids for homologous recombination for insertion of the mutagenized genes of phaB1, phaB2, or had-crt2 into chromosome 1 of R. eutropha at the phaB1 locus were constructed from pK18mobsacB-AR previously made for deletion of phaB1 [33 (link)]. The target genes were individually inserted into pK18mobsacB-AR as located downstream of phaA with the same orientation.
pColdII-phaB1, pColdII-phaB2, and pET15b-phaB3 vectors for overproduction of N-His6-tagged PhaB1, PhaB2, and PhaB3 in E. coli, respectively, were constructed as described in Supporting Information. Site-directed mutagenesis of phaB1 was carried out by QuickChange protocol. A DNA fragment consisting of a tandem of had and crt2 was prepared by fusion PCR. Several plasmids were constructed by blunt-end ligation of a DNA fragment with a linear fragment of the backbone plasmid prepared by inverse PCR. pBPP-ccrMeJAc-emd was constructed by replacement of phaJ4a in pBPP-ccrMeJ4a-emd [23 (link), 44 (link)] by phaJAc amplified with pEE32 [10 (link)] as a template. Plasmids for homologous recombination for insertion of the mutagenized genes of phaB1, phaB2, or had-crt2 into chromosome 1 of R. eutropha at the phaB1 locus were constructed from pK18mobsacB-AR previously made for deletion of phaB1 [33 (link)]. The target genes were individually inserted into pK18mobsacB-AR as located downstream of phaA with the same orientation.
5
PCR Amplicon Denaturation and T7 Endonuclease I Assay
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PCR reactions to amplify specific on-target or off-target sites were performed using KOD-Plus-ver.2 DNA polymerase (Toyobo) in accordance with the manufacturer’s protocol. The resulting PCR amplicons were denatured and re-annealed in 1× NEB buffer 2 (NEB) in a total volume of 9 μl under the following conditions: 95 °C for 5 min, reduction from 95 °C to 25 °C at a rate of −0.1 °C s−1 and indefinite incubation at 4 °C. After re-annealing had been performed, 1 μl of T7 endonuclease I (NEB, 10 U μl−1) was added and the product was incubated at 37 °C for 15 min.
6
Biochemical Characterization of Enzymes
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Amino acids and pyruvic acid were purchased from Wako Pure Chemicals, Co., Ltd. (Japan), Watanabe Chemical Industries, Ltd. (Japan) or Sigma Japan. Restriction enzymes were from New England Biolabs Japan. KOD -plus ver. 2 DNA polymerase was from Toyobo, Co., Ltd. (Japan). Methanol and acetonitrile were from Kanto Kagaku, Co., Ltd. (Japan). Molecular weight standards for gel filtration chromatography were from GE Healthcare Japan. All other reagents were of analytical or molecular biology grade.
7
Molecular Cloning of GcvB Mutants
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The oligonucleotides and plasmids used in this study are listed in Tables S4 and S5 , respectively. The E. coli GcvB expression plasmid (pPL‐gcvB) was constructed by cloning the XbaI‐digested PCR fragment amplified with 5′‐end phosphorylated JVO‐0237 and MMO‐0086 into the pPL vector with an Ap resistance marker and a p15A ori to express GcvB from a constitutive promoter. Expression plasmids of GcvB deletion mutants were constructed via PCR amplification from the original GcvB expression plasmid using KOD plus ver.2 DNA polymerase (Toyobo), DpnI digestion of the template plasmid, and self‐ligation of purified PCR products. The plasmid‐borne GcvB was mutated by site‐directed mutagenesis via PCR amplification using overlapping primers (Tables S4 ). The purified PCR products were directly transformed into E. coli DH5α strain after DpnI digestion of the template plasmid. The sequences of the GcvB mutants are shown in Table S6 . Translational fusions were constructed as previously described (Corcoran et al., 2012 (link); Urban & Vogel, 2007 (link)). The detailed characteristics of reporter plasmid constructions are listed in Table S7 , and the inserts of all translational fusions are listed in Table S8 . The pSydeE plasmid was constructed by PCR amplification with pSydeE‐5′ and pSydeE‐3′, digestion with EcoRI and BamHI, and cloning into the pSTV28 vector (Takara Bio).
8
Rapid Amplification of cDNA Ends for SaERF1 Sequencing
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Because no full-length clone of the SaERF1 cDNA sequence was available, we obtained the 3′-end sequence of SaERF1 by RACE-PCR from S1 leaf total RNA using a first-choice RLM RACE Kit (Ambion, Foster City, CA, USA) according to the manufacturer’s protocol.
To determine the sequence of the 5′-end of the SaERF1 cDNA, genomic DNA from S1 leaves was digested with Xba I and Xho I, and then circularized with T4 DNA ligase (Takara Bio, Kusatsu, Japan). Nested inverse PCR was performed with circularized genomic DNA as the template, using KOD -Plus- Ver.2 DNA polymerase (Toyobo, Osaka, Japan) with a pair of primers that introduced restriction enzyme cleavage sites. PCR protocol: 35 cycles of 5 s at 94 °C, 1 s at 55 °C, and 60 s at 68 °C. The resultant PCR products were subcloned and sequenced.
Finally, the ORFs of SaERF1 and SaERF2 cDNAs were amplified using total RNA from S1 and S2 leaves as cDNA template, respectively, using ReverTra-Plus-TM and KOD -Plus- Ver.2. Sequence data can be found in the GenBank/EMBL data libraries under accession numbers LC424188 and LC424189 for SaERF1 and SaERF2, respectively.
To determine the sequence of the 5′-end of the SaERF1 cDNA, genomic DNA from S1 leaves was digested with Xba I and Xho I, and then circularized with T4 DNA ligase (Takara Bio, Kusatsu, Japan). Nested inverse PCR was performed with circularized genomic DNA as the template, using KOD -Plus- Ver.2 DNA polymerase (Toyobo, Osaka, Japan) with a pair of primers that introduced restriction enzyme cleavage sites. PCR protocol: 35 cycles of 5 s at 94 °C, 1 s at 55 °C, and 60 s at 68 °C. The resultant PCR products were subcloned and sequenced.
Finally, the ORFs of SaERF1 and SaERF2 cDNAs were amplified using total RNA from S1 and S2 leaves as cDNA template, respectively, using ReverTra-Plus-TM and KOD -Plus- Ver.2. Sequence data can be found in the GenBank/EMBL data libraries under accession numbers LC424188 and LC424189 for SaERF1 and SaERF2, respectively.
9
Standard Molecular Cloning Procedures
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DNA manipulation was carried out by standard procedures, and restriction endonucleases and DNA modification enzymes were purchased from Takara Bio (Otsu, Shiga, Japan) or Toyobo (Osaka, Japan), unless otherwise noted. PCR reactions for gene cloning were carried out with KOD-Plus-ver.2 DNA polymerase (Toyobo), and those for other purposes such as colony PCR were done with KOD FX DNA polymerase (Toyobo). Supplementary Table S2 lists the sequences of oligonucleotide primers used in this study.
10
Plasmid-based DNA Amplification Protocol
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KOD-Plus-Ver.2
DNA polymerase (Toyobo, Osaka, Japan) was applied to synthesize dsDNAs
(PCR amplicons) using pEGFP-C1 (Clontech, Mountain View, CA) plasmid
as a template. PCR products underwent agarose electrophoresis and
gel purification to remove the template and free dNTPs (using Universal
DNA purification kit, Tiangen, Beijing, China). The amount of PCR
products was determined by the OD 260 value. The sequences of PCR
primers and dsDNAs are present inSupporting Table 5 and Supporting Materials , respectively.
DNA polymerase (Toyobo, Osaka, Japan) was applied to synthesize dsDNAs
(PCR amplicons) using pEGFP-C1 (Clontech, Mountain View, CA) plasmid
as a template. PCR products underwent agarose electrophoresis and
gel purification to remove the template and free dNTPs (using Universal
DNA purification kit, Tiangen, Beijing, China). The amount of PCR
products was determined by the OD 260 value. The sequences of PCR
primers and dsDNAs are present in
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