RT-PCR was performed to amplify cDNAs encoding medaka Cdk1 (303 residues), Cdk9 (393 residues), Ccni (342 residues), and Ribosomal protein L7 (Rpl7; 245 residues) using KOD Neo DNA polymerase (Toyobo, Tokyo, Japan) with ovary cDNA. The primers used are listed in Supplementary Table S1 . The amplified products were phosphorylated and inserted into the prokaryotic expression vector pET30a (Novagen, Madison, WI, USA), which had been previously digested by EcoRV. Expression, purification, and dialysis of the protein were performed according to previously published methods [15 (link)]. Anti-medaka Cdk1, Cdk9, Ccni, and Rpl7 antibodies were generated using mice according to methods described previously [14 (link)]. Mouse anti-medaka Pgr antibody [16 (link)], rat anti-medaka Pgr antibody [16 (link)], and rabbit anti-medaka Mmp15 antibody [6 (link)] were prepared as described previously. Antibodies were purified as previously described [7 (link)], and the resulting purified antibodies were used in the experiments.
Kod neo dna polymerase
Manufactured by Toyobo
Sourced in Japan
KOD Neo DNA polymerase is a high-fidelity thermostable DNA polymerase derived from the hyperthermophilic archaeon Thermococcus kodakaraensis. It possesses 3'→5' exonuclease proofreading activity, which provides enhanced fidelity during DNA amplification.
Automatically generated - may contain errors
4 protocols using kod neo dna polymerase
1
Medaka Cdk1, Cdk9, Ccni, and Rpl7 Protein Expression
2
Genomic DNA Extraction and Sequencing
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Fresh leaves were collected from the field-grown plants for a genomic DNA extraction with the cetyltrimethylammonium bromide method (Murray and Thompson 1980 (link)). The primers used to amplify the promoter, 5′ UTR, exon, intron, and 3′ UTR were designed based on the Nipponbare GNP1 and NAL1 alleles. The PCR was completed with KOD NEO DNA polymerase (Toyobo, Shiga, Japan) and a standard PCR protocol.
Because of the existence of heterozygous genotypes in O. rufipogon, the PCR product was ligated into the pGEM-T Easy Vector (Promega, USA) and then sequenced, after which one allele sequence was randomly selected. To ensure accuracy, the sequencing was performed twice with the ABI 3730 system at the Beijing Genomics Institute, China. Sequence contigs were assembled with the SEQUENCHER 4.1.2 program (Gene Codes Corporation, Ann Arbor, MI, USA). Details regarding the PCR primers and sequencing are listed in Additional file14 : Table S6.
Because of the existence of heterozygous genotypes in O. rufipogon, the PCR product was ligated into the pGEM-T Easy Vector (Promega, USA) and then sequenced, after which one allele sequence was randomly selected. To ensure accuracy, the sequencing was performed twice with the ABI 3730 system at the Beijing Genomics Institute, China. Sequence contigs were assembled with the SEQUENCHER 4.1.2 program (Gene Codes Corporation, Ann Arbor, MI, USA). Details regarding the PCR primers and sequencing are listed in Additional file
3
Cloning and Expression of PpMYB10 Transcription Factors
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ORFs of PpMYB10.1/2/3 were generated from first-strand cDNA of ‘Akatsuki’ by using PCR with KOD-Neo DNA polymerase (Toyobo, Osaka, OSK, Japan). Purified PCR fragments with the expected size bands were cloned into the pENTR/D-TOPO vector (Invitrogen), according to the manufacturer’s instructions. Subsequently, the ORFs of PpMYB10.1/2/3 were inserted into the pGWB2 vector under transcriptional control of the 35S CaMV promoter [33 (link)] by using LR clonase (Invitrogen). Recombinant pGWB2 plasmids were then transferred into Agrobacterium tumefaciens LBA4404 by using the heat-shock method and grown at 28 °C on Luria-Bertani medium with kanamycin, hygromycin, and rifampicin.
4
Cloning, Expression, and Antibody Production
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A 858-bp cDNA coding for the complete Cebpb protein (286 residues) was amplified by RT-PCR using KOD Neo DNA polymerase (Toyobo, Tokyo, Japan) with ovary cDNA.
The amplified product was phosphorylated and ligated into the vector pET30a (Novagen, Madison, WI), which had been previously digested by EcoRV. The protein antigen was produced in the bacterial expression system. Expression, purification, and dialysis of the protein were performed as previously described (Ogiwara and Takahashi, 2007) (link). Anti-medaka Cebpb antibody was generated using mice according to the method previously described (Ogiwara et al., 2013) (link). Recombinant Pgr was produced as described previously (Hagiwara et al., 2014) (link) and used to immunize rats to obtain anti-medaka Pgr antibody. Mouse anti-medaka Pgr antibody (Hagiwara et al., 2014) (link), rabbit anti-medaka Mmp15 antibody (Ogiwara and Takahashi, 2007) (link), and rabbit anti-medaka Actb antibody (Ogiwara et al., 2012) (link) were prepared as described previously.
Antibodies were purified using an Immobilon polyvinylidene difluoride (PVDF) (Millipore, Bedford, MA) membrane as described (Ogiwara et al., 2012) (link), and the resulting purified antibodies were used for the experiments.
The amplified product was phosphorylated and ligated into the vector pET30a (Novagen, Madison, WI), which had been previously digested by EcoRV. The protein antigen was produced in the bacterial expression system. Expression, purification, and dialysis of the protein were performed as previously described (Ogiwara and Takahashi, 2007) (link). Anti-medaka Cebpb antibody was generated using mice according to the method previously described (Ogiwara et al., 2013) (link). Recombinant Pgr was produced as described previously (Hagiwara et al., 2014) (link) and used to immunize rats to obtain anti-medaka Pgr antibody. Mouse anti-medaka Pgr antibody (Hagiwara et al., 2014) (link), rabbit anti-medaka Mmp15 antibody (Ogiwara and Takahashi, 2007) (link), and rabbit anti-medaka Actb antibody (Ogiwara et al., 2012) (link) were prepared as described previously.
Antibodies were purified using an Immobilon polyvinylidene difluoride (PVDF) (Millipore, Bedford, MA) membrane as described (Ogiwara et al., 2012) (link), and the resulting purified antibodies were used for the experiments.
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