Primstar max dna polymerase

To isolate GtMIF1-1 and GtMIF1-2, total RNAs were extracted from flowers of F₂ line plants that showed lower L^* values, using an RNeasy Plant Mini Kit (Qiagen). For synthesising first-strand cDNA, 100 ng of total RNA was reverse transcribed using the ReverTra Ace qPCR RT Master Mix with gDNA Remover kit (Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer’s instructions. The open reading frame (ORF) of GtMIF1-1 and GtMIF1-2 was amplified by PCR using PrimSTAR max DNA Polymerase (Takara Bio Inc., Shiga, Japan) and synthesised cDNA as a template. The DNA fragments were cloned into pDONR221, and the sequences were confirmed. Primer sets for cloning are shown in Supplementary Table S3.

PPP1r18-myc was subcloned to pQCIXN vector (Takara bio) with PCR methods using PrimSTAR Max DNA polymerase (Takara bio). Control or hPPP1r18-myc vector were transfected in Platinum-E Retroviral Packaging Cell Line, Ecotropic (Plat-E) cells (Cell Biolabs, Inc., CA) and incubated for 1 day. The supernatant of the retrovirus production cells was moved to RetroNectin coated dishes and incubated for 6 h to make retrovirus coating dish.

The mgsA genes from Clostridioides difficile 630 (Genbank ID: AJP10844.1) and Oceanithermus profundus DSM 14,977 (Genbank ID: ADR35584.1) encoding the MGS enzymes were synthesized by Bioneer Co. (Republic of Korea). For affinity chromatography-mediated protein purification, a Strep tag consisting of a nine residue-long polypeptide was cloned via PCR to the N- or C-terminal of the MGSs with specific primers (Supplementary Table S1). A six-aspartate tag was also added to the N-terminal of opMGS to increase the solubility of the protein. PrimSTAR Max DNA polymerase (Takara, Japan) was used for the PCR following the manufacturer’s protocol. The pETduet-1 vector was linearized with QuickCut NdeI & KpnI restriction enzymes (Takara, Japan) before ligation with the PCR products using an In-Fusion HD Cloning Kit (Clontech, USA). The constructed vectors were transformed into competent E. coli DH5α (RBC, Taiwan) and selected on LB agar plates supplemented with 100 µg ml⁻¹ ampicillin as a marker. The cells were grown in 50 ml LB medium supplemented with 100 μg ml⁻¹ ampicillin at 37 °C, 200 rpm in a shaking incubator. The vectors were mini-prepped and their sequences were confirmed by DNA sequencing.

The promoter and full length of the bla_NDM genes were amplified with primers NDM-F-EcoRI (5′-CCGGAATTCTTGAAACTGTCGCACCTCAT-3′) and NDM-R-XbaI (5′-CTAGTCTAGAACGCCTCTGTCACATCGAA-3′) using PrimSTAR Max DNA Polymerase (Takara, China). After restriction enzyme digestion, the PCR products were ligated to the vector PET28a to generate PET28a-NDM-5, PET28a-NDM-36, PET28a-NDM-37 respectively. The correct constructs were confirmed by Sanger sequencing, followed by transformation into E. coli DH5α. Antimicrobial susceptibilities of these constructs were determined as described above. The empty pET28a plasmid was used as a control.

E. coli strain DH5α was used for gene cloning, and the Rosetta2(DE3)pLysS strain was used for recombinant protein expression. The expression vector pET28a was used to express the recombinant proteins. P. yayanosii genomic DNA was extracted from P. yayanosii cultures in our lab. PrimSTAR Max DNA polymerase was purchased from Takara (Shiga, Japan). Nickel–nitrilotriacetic acid resin was purchased from Bio-Rad (Hercules, CA, USA). Adenosine 3′,5′-bisphosphate (pAp) was purchased from Sigma (St Louis, MO, USA). The Cycle Pure Kit was purchased from Omega (Guangzhou, China). Methanol, formic acid, and ammonium formate were purchased from Aladdin (Shanghai, China) for the HPLC assay.