Kod plus high fidelity dna polymerase

C. elegans was maintained using standard procedures^{48 (link)}. unc-33(ky880); kyIs445 and Pdes-2::unc-33L were described previously^{13 (link)}. Mutations were introduced to the Pdes-2::unc-33L plasmid by PCR-based mutagenesis with KOD-plus high fidelity DNA polymerase (TOYOBO, Tokyo, Japan). Injections were performed as described^{49 (link)}. In short, wild type and mutant unc-33 plasmids (10 ng/μl) and Podr-1::DsRed (50 ng/μl) were injected to unc-33(ky880); KyIs445 mutant worms. DsRed positive worms were transferred to new plates and stable transmission of the extrachromosomal array was determined. For rescue experiments, only DsRed positive worms were observed and analyzed by LSM710 confocal microscope (Carl Zeiss).
For the statistical analysis of the dendritic phenotype, worms were observed in a blind fashion. The observer did not know genotypes until the end of the test. Each worm was classified as “wild type” or “unc-33” by the synaptic vesicle localization. 100 worms were observed for each genotype and plotted as bar graphs. For the quantification of axonal fluorescent intensity, worms were observed in the same manner as the dendritic analysis. The mean fluorescent intensity was measured using Image J (NIH). A 100-μm region of the axon was selected randomly to measure the mean fluorescent intensity.

Expression plasmids for transgenic worm lines were made using the pSM vector (C. Bargmann), a derivative of pPD49.26 (A. Fire). The mig-13 promoter was cloned between SphI/AscI, and C.elegans unc-43 isoform d or human CAMK2α was cloned between NheI/KpnI or AscI/NheI, respectively. P.H466Y and p.H477Y mutations were introduced by PCR-based mutagenesis using KOD-plus- high fidelity DNA polymerase (TOYOBO, Tokyo, Japan). Transgenic worms were generated as described (Mello, 1995 (link)). Plasmids were injected into animals at 10 ng/μl (in the case of Pmig-13::unc-43) and 50 ng/μl (in the case of Pmig-13::CAMK2α) together with coinjection markers at 90 ng/μl.

All chemicals were purchased from Nacalai Tesque (Tokyo, Japan) or Wako Pure Chemical (Tokyo, Japan) unless otherwise specified. KOD Plus high-fidelity DNA polymerase was purchased from Toyobo (Tokyo, Japan). Restriction enzymes and the DNA ligation kits used were from Takara (Shiga, Japan).

5′RACE was performed according to the method described in Bensing et al. (1996) (link). Briefly, 6 μg of total RNA was treated with 75 U of tobacco pyrophosphatase (TAP; Nippon Gene) at 37°C for 30 min in the presence of 20 U of RNaseOUT. The TAP-treated and -untreated RNA samples were mixed with the RNA oligonucleotide (5′-AUAUGCGCG AAUUCCUGUAGCUAGAAGAAA-3′) and ligated by 40 U of T4 RNA ligase (TAKARA Bio) at 16°C overnight. The ligated RNA samples were mixed with 1 pmol of gene-specific primer traJ-R2 (5′-TCTCTTCGATCTTCGCCAGC-3′) and reverse transcribed by 100 U of SuperScriptIII at 50°C for 60 min in the presence of 20 U of RNaseOUT. The cDNA fragment spanning the ligated RNA oligonucleotide and the 5′ end of traJ transcript was amplified by KOD-Plus high-fidelity DNA polymerase (TOYOBO) using primers Oligo-F1 (5′-TATGCGCGAATTCCTGTAGC-3′) and traJ-R. The amplified fragment was cloned into HincII-digested pBluescript II SK(-) vector (Stratagene), and the inserts from several clones were sequenced using M13 primers.

To substitute the arginine residue at position 566 (Arg-566) of ASP to the alanine residue, we site-directly mutated asp on pSA19-5528 according to the polymerase chain reaction (PCR)-based one-step mutagenesis method [20 (link),21 (link)]. The PCR was carried out using KOD plus high-fidelity DNA polymerase (Toyobo, Osaka, Japan). The primers 5′-ctctctgctcaatgccgagacccgtgag-3′ and 5′- ctcacgggtctcggcattgagcagagag-3′ were used for mutation of the target gene. The mutation was verified by DNA sequencing. The plasmid thus obtained was designated pSA19-5528[R566A].

Human SUV420H1 and SUV420H2 cDNAs were amplified from HBL-100 cDNA and inserted into the XhoI/BamHI sites and EcoRI/BamHI sites of vector pEGFP-C1, respectively. PCR reactions were performed using KOD Plus high-fidelity DNA polymerase (Toyobo, Osaka, Japan). All cDNA constructs were verified by DNA sequencing.