High fidelity dna polymerase

The names and sequences of primers used for amplification of the PpMYB36 genomic sequence are given in Supplementary Table 5. DNA fragments were amplified using high-fidelity DNA polymerase (Takara) using reaction conditions recommended by the manufacturer. The PCR products were purified and cloned into the PMD 19-T vector (Takara). Nucleotide sequences of 10 independent clones of each fragment per sample were determined. BLAST analysis of the amino acid sequence of PpMYB36 was performed in the Arabidopsis information resource,⁵ and a phylogenetic tree was constructed using MEGA 5.2 (Tamura et al., 2011 (link)). Promoter sequence analysis was performed using the PlantCARE online database.⁶

The putative effectors were selected from H. avenae transcript libraries. Signal peptides were predicted using signalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/)68 (link), and putative transmembrane helices were predicted by TMHMM Server v.2.0
(http://www.cbs.dtu.dk/services/TMHMM/). The open reading frames were predicted by ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/gorf.html). The coding sequences of candidate genes were amplified with high-fidelity DNA polymerase (TaKaRa, Japan). Then, the coding sequences were cloned into the plant expression vector pYBA1143 using restriction enzyme sites. The HaEXPB2 coding sequences with and without signal peptide were amplified using gene-specific primers containing BamHI and HindIII restriction enzyme sites. The HaEXPB2Δ260-289 sequence was amplified using HaEXPB2F (BamHI)/HaEXPB2R1 (Hind III). HaEXPB2Δ117-289 sequence was amplified using HaEXPB2F (BamHI)/HaEXPB2R3 (Hind III). The sequence of HaEXPB2ΔCBD, HaEXPB2ΔDPBB, and HaEXPB2Δ117-232 were amplified using HaEXPB2-4R/HaEXPB2-4F, HaEXPB2-5R/HaEXPB2-5F, HaEXPB2-6R/HaEXPB2-6F and HaEXPB2F (BamHI)/HaEXPB2R1 (Hind III) by overlapping PCR, respectively (Supplementary Table 2). The resulting amplified fragments were cloned into the respective sites in the modified pYBA1143 vector, which contained an HA tag. All constructs in this study were confirmed by sequencing.

SNHG1 cDNA fragments were amplified from the SNHG1 plasmid as templates by utilizing high fidelity DNA polymerase (Takara, Kyoto, Japan). Based on this template, fluorescein-labeled lncTCF7 FISH probe DNA was prepared with fluorescein-12-dUTP (Roche, Mannheim, Germany) and Klenow DNA polymerase (Vazyme, Nanjing, China) as per the manufacturer’s protocol. Then, 4-µm frozen sections were made from bladder cancer tissues and adjacent normal tissues. Subsequent to 5-min immersion in proteinase K (MCE, NJ, USA), the slides were washed twice in 2× saline sodium citrate (SSC) (Merck KGaA, Darmstadt, Germany). The FISH hybridization solution encompassing 30 ng/µL lncTCF7 FISH probe DNA (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China) was dripped onto the tissue sections before 16-h incubation at 37 °C. The slides were then washed in 0.4 × SSC/0.001% NP-40 (Merck KGaA, Darmstadt, Germany) for 5 min at 56 °C, followed by another 2-min washing in 0.4 × SSC/0.001% NP-40. After being dripped with 4′,6-Diamidino-2-Phenylindole (DAPI)-encompassing sealing agent, the slide was mounted and observed under a fluorescence microscope (Olympus, Tokyo, Japan).