Hieff pcr master mix

Pichia pastoris GS115 was purchased from Invitrogen (Carlsbad, CA, United States), and M. lysodeikticus (CGMCC 1.4547) cells were purchased from China General Microbiological Culture Collection Center. The template plasmids p905M, pGAPZB, pGAPZM, and pGAPZN were generated in our laboratory. All the media, including the Luria–Bertani media (LB), Yeast Extract Peptone Dextrose media (YPD), buffered glycerol-complex media (BMGY), buffered methanol-complex media (BMMY), minimal dextrose media (MD), and Skerman’s basal mineral salt media (BSM) were prepared based on the Invitrogen PichiaPink^TM Expression System manual¹.
Zeocin antibiotics, PageRuler^TM Prestained Protein Ladder and T4 DNA ligase were purchased from Thermo Fisher Scientific (Waltham, MA, United States). 5,5′-Dithiobis-2-nitrobenzoic acid (DTNB), N-acetyl-L-cysteine, Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), and Hieff^TM PCR Master Mix were purchased from Yeasen (Shanghai, China). All other restriction enzymes were purchased from New England Biolab (Ipswick, MA, United States).

RNA was extracted from the different cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. After lysis with TRIzol, chloroform was added to the samples and the samples were centrifuged at 12,000 × g for 15 min. An equal volume of isopropanol was added to the upper transparent RNA-containing solution. After centrifugation, the RNA pellet was washed twice with 75% ethanol. RNA was reverse transcribed into cDNA using PrimeScript™ RT Master Mix (Takara, RR036A). Real-time PCR was performed using HieffTM PCR Master Mix (Yeasen) according to the manufacturer’s instructions, and the relative expression of target genes was normalized to that of GAPDH.

Conventional PCR was performed with a standard program in 20.0 μL reaction volume on a PCR cycler (Eppendorf, Germany). The composition of the reaction mixture contained 10.0 μL of 2 × Hieff™ PCR Master Mix (Yeasen Biotech Co., Ltd.), 1.0 μL of template, 0.5 μM of each primer, and 8 μL of ddH₂O. Cycle times were as follows: 95°C for 5 min (initial denaturation), 40 cycles of 95°C for 30 s (denaturation), 54°C for 30 s (annealing), and 72°C for 20 s (extension), followed by a final step of 10 min at 72°C (extension). The PCR products were detected by 1.5% agarose gel electrophoresis. Three standard plasmids were used as positive control, and ddH₂O played the role of the negative control.

Total RNA was isolated from the HEK293T cells using TRIzol reagent (Thermo Fisher Scientific, Cat#15596026) and treated with RNase-free DNase (Takara, Dalian, China, EN0521) according to the manufacturer’s instructions. The quality of RNA was examined using the electrophoresis on 1% agarose gel, and the purity of RNA was verified on the basis of the ratio of OD260/280 on NanoDrop One (Thermo Scientific). Two μg of total RNA was used as a template to synthesize cDNA using the cDNA synthesis kit (Thermo Fisher Scientific, Cat#K1622). The cDNAs were subjected to the real-time quantitative PCR (qPCR), which were performed on a 96™ Real Time PCR Detection System (Applied Biosystems™ 7500), in 10 μL reaction mixtures containing 5 μL SYBR^® TB Green^® Premix Ex Taq™ (Tli RNaseH Plus) (Takara, Dalian, China, Cat#RR420A). The thermal profile consists of 30 s at 95°C, followed by 40 cycles of 5 s at 95°C and 5 s at 60°C. The 2^–ΔΔCT method was used to calculate the relative gene expression values. The details for the design of primers and qPCR conditions were described as previously (Jin et al., 2021 (link)). The RT-PCR was performed with 2 × Hieff^® PCR Master Mix (Yeasen, Shanghai, China, 10102ES08), extension time of PCR program is 1 min, and PCR products were examined in the 1.5% agarose gel after DNA electrophoresis.

RNA was extracted using RNAprep Pure Plant Plus Kit (TIANGEN, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized using Takara PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Japan). PCR was performed using Hieff™ PCR Master Mix (Yeasen, China). Quantitative RT-PCR was performed on a LightCycler 480 System (Roche, Germany) using SYBR Premix Ex-Taq (TaKaRa, Japan) according to the manufacturer’s protocols. Primers used in the assays are listed in Table S1.

Total RNA of B. distachyon was extracted using TRIzol Reagent (Ambion, Waltham, MA, USA), and purified with an EasyPure Plant RNA Kit (TianGen, Beijing, China). Subsequently, cDNA first-strand synthesis was conducted by reverse transcription using a NovoScript Plus All-in-one SuperMix kit (Novoprotein, Shanghai, China). Specific primers for RT-PCR and qRT-PCR are listed in Supplementary Table S8. The roots, stems, leaves, and seeds were sampled at the same time intervals. To investigate the expression of BdGATA genes in different tissues/organs, we carried out the method for tissue sample collection as previously described [46 (link)]. RT-PCR was conducted using a MonScript RTase II kit (Monad, Suzhou, China). PCR was performed with 10–15 pmol of each primer, DNA templates (60 ng), double-distilled H₂O, and Hieff PCR Master Mix (Yeasen, Shanghai, China) in a total volume of 20 μL. qRT-PCR was performed using a TransStart Tip Green qPCR SuperMix kit (TransGen, Beijing, China) in a CFX 96 qPCR instrument (BioRad, Hercules, CA, USA). BdUBC (Bradi4g00660) was amplified as the normalization control.