Primestar hs dna polymerase

Cocoa genes encoding GPATs and LPATs were amplified using primers described in Additional file 1: Table S1 from cocoa fruit cDNA using the PrimeSTAR HS DNA polymerase (Takara) according to the manufacturer’s instruction. The one GPAT and one LPAT sequences amplified from cocoa cDNA, which were different from the available annotated genes, were deposited at the GenBank database under the accession numbers MF352000-MF352001. The primers used to amplify cocoa genes, promoters and terminators are listed in Additional file 1: Table S1 and some primers used for cocoa gene combination expression were the same as described before [9 (link)]. The LsGPAT gene was cloned from L. starkeyi cDNA using PrimeSTAR HS DNA polymerase (Takara) and the sequence was the same as described before [26 (link)].
The cocoa gene expression cassettes were verified by sequencing and illustrated in Fig. 2. Gibson assembly (NEB) was used to construct cocoa gene expression plasmids by ligation of the gene expression cassettes and the amplified linear backbone fragment of plasmid pBS01A, and were further verified by PCR and Sanger sequencing (Additional file 1: Table S2). The constructed plasmids were used to transform S. cerevisiae to construct the strains listed in Table 1.Fig. 2

Schematic organization of cocoa gene expression cassettes in each of the expression plasmids

Total cellular DNA (500 ng) extracted from fibroblasts by using a Puregene Core Kit A (Qiagen, USA) was used as template for each PCR reaction. Amplicons generated with eight sets of primers were produced with Takara PrimeSTAR HS DNA polymerase (Takara Bio, Japan) in a reaction volume of 50 μl containing 2.5 mM dNTP mixture, 0.3 μM forward primer, 0.3 μM reverse primer, and PrimeSTAR HS DNA polymerase. The sequences of the primers are shown in Supplementary Table 2. The cycling conditions used were as follows: 1 cycle of 98 °C for 1 min; then 16 cycles of 98 °C for 10 s, 55 °C for 15 s, and 72 °C for 3 min. PCR amplicons were purified with a QIAquick PCR purification kit (Qiagen). Purified DNA was subjected to deep sequencing with a Genome Analyzer IIx (Illumina, USA). Sequencing reads were assembled and compared with the reference sequences (GenBank accession no. AB055387).

Mammalian constructs encoding Myc-tagged wild-type LRRK2, its kinase-dead counterpart and two PD-associated pathogenic LRRK2 mutants (pcDNA3.1-Myc-LRRK2-WT, -D1994A, -G2019S and -R1441C, respectively) were generated, as described elsewhere (Shin et al., 2008 (link); Heo et al., 2010 (link)). The mammalian expression vector for HA-tagged human wild-type RCAN1 (RCAN1-1S) was a kind gift from S. de la Luna (Genomics Regulation Center, Barcelona, Spain). Mammalian expression vectors encoding HA-tagged RCAN1 deletion mutants (HA-RCAN1^1–95, HA-RCAN1^1–125, HA-RCAN1^30–197, and HA-RCAN1^90–197) were constructed, as previously described (Lee et al., 2012 (link)). Bacterial expression vectors encoding GST-fused wild-type RCAN1 (pGEX4T-1-GST-RCAN1) and five deletion mutants (RCAN1^1–95, RCAN1^1–125, RCAN1^1–145, RCAN1^1–160 and RCAN1^1–175) were produced by PCR amplification of wild-type RCAN1 and its deletion mutants using Prime STAR-HS DNA Polymerase (TAKARA, Shiga, Japan) and sub-cloning into the pGEX4T-1 vector. Bacterial constructs encoding GST-fused RCAN1 mutants having a substitution at Ser149, Thr151, Thr152, Thr154, or Ser162 with alanine, respectively, were generated through PCR amplification of pGEX4T-1-GST-RCAN1-WT as a template. Site-directed mutagenesis reactions were performed using the Prime^®STAR HS DNA polymerase (Takara Bio Inc., Shiga, Japan).