PrimeSTAR GXL DNA Polymerase, QuickCut Enzyme, Alkaline Phosphatase, and T4 DNA Ligase were purchased from Takara (Dalian, China). Bacterial genomic DNA was extracted using a TIANamp Bacteria DNA Kit (TIANGEN, Beijing, China). DNA was purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA, USA). All positive clones were verified by PCR (Additional file 1 : Figs. S2, S3, S4).
Quickcut enzyme
Manufactured by Takara Bio
Sourced in China
QuickCut enzymes are a suite of molecular biology tools designed for precise DNA manipulation. They enable efficient and reliable DNA cleavage, facilitating various DNA engineering workflows. The core function of QuickCut enzymes is to provide accurate and reproducible DNA digestion for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by Takara Bio (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Primestar gxl dna polymerase, by Takara Bio (1 mentions)
Zymoclean gel dna recovery kit, by Zymo Research (1 mentions)
Tianamp bacteria dna kit, by Tiangen Biotech (1 mentions)
Cocl2 6h2o, by Merck Group (1 mentions)
Protein a sepharose beads, by Santa Cruz Biotechnology (1 mentions)
Protein g sepharose beads, by Santa Cruz Biotechnology (1 mentions)
17 aag, by Merck Group (1 mentions)
Primestar hs premix, by Takara Bio (1 mentions)
Dna loading buffer, by Takara Bio (1 mentions)
Dna marker, by Takara Bio (1 mentions)
Hindiii, by New England Biolabs (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Bortezomib, by Selleck Chemicals (1 mentions)
Plus reagent, by Thermo Fisher Scientific (1 mentions)
β actin, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Glycogen, by Merck Group (1 mentions)
6 protocols using quickcut enzyme
1
Molecular Cloning with Takara Enzymes
2
Phospho-AKT Signaling Pathway Analysis
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Phospho-AKT, polyclonal antibodies against AKT and TNF-α, and the monoclonal antibody against S6 were from Cell Signaling Technology (Beverly, MA). Monoclonal mouse antibodies against cyclin D1, IL6, and HSP90 (Santa Cruz Biotechnology, CA), GAPDH (Abcam Biotechnology, Cambridge, MA), β-actin (Sigma-Aldrich, St. Louis, MO), and His (Quanshijin Biotechnology, Beijing, China) were also used.
Protein A-sepharose beads and protein G-sepharose beads were purchased from Santa Cruz Biotechnology. Bortezomib was obtained from Selleck (Shanghai, China), and 17-AAG and CoCl2·6H2O were obtained from Sigma-Aldrich. Lipofectamine and PLUS reagent were purchased from Invitrogen Life Technologies. The wild-type prokaryotic IDH2 expression vector (EX-C0462-B01) was from FulenGen Co., Ltd. (Guangzhou, China). The empty eukaryotic vector pCMV6-AC-Myc-His was purchased from Origene Technologies (Rockville, MD). The restriction endonucleases HindIII and MluI were obtained from New England Biolabs (MA). Premix PrimeSTAR HS, QuickCut enzyme, T4 DNA ligase, DNA Marker, and DNA Loading Buffer were purchased from Takara (Kusatsu, Japan). Primer synthesis (TableS1 ) and sequencing were completed by Sangon Biotech (Shanghai, China).
Protein A-sepharose beads and protein G-sepharose beads were purchased from Santa Cruz Biotechnology. Bortezomib was obtained from Selleck (Shanghai, China), and 17-AAG and CoCl2·6H2O were obtained from Sigma-Aldrich. Lipofectamine and PLUS reagent were purchased from Invitrogen Life Technologies. The wild-type prokaryotic IDH2 expression vector (EX-C0462-B01) was from FulenGen Co., Ltd. (Guangzhou, China). The empty eukaryotic vector pCMV6-AC-Myc-His was purchased from Origene Technologies (Rockville, MD). The restriction endonucleases HindIII and MluI were obtained from New England Biolabs (MA). Premix PrimeSTAR HS, QuickCut enzyme, T4 DNA ligase, DNA Marker, and DNA Loading Buffer were purchased from Takara (Kusatsu, Japan). Primer synthesis (Table
3
Detecting PmMYB7 Autoactivation Using Yeast Two-Hybrid
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To detect autoactivation of the PmMYB7 gene, we used the pGBKT7 vector to construct a recombinant plasmid as a bait vector. The pGBKT7 vector was digested at the EcoRI and XbaI sites using QuickCut enzymes (Takara). The PmMYB7 coding sequence was inserted into the pGBKT7 vector. Approximately 100 ng of pGBKT7-PmMYB7 bait vector and 100 ng of pGADT7 prey vector plasmids were cotransformed into S. cerevisiae AH109 cells to assess autoactivation and toxicity. pGBKT7-p53 and pGADT7-largeT were cotransformed as positive controls, and pGBKT7-laminC and pGADT7-largeT were cotransformed as negative controls. The transformed competent cells were cultured on SD/-Leu/-Trp (DDO) and SD/-Trp/-Leu/-His/-Ade (QDO) plates at 30 °C for 3–5 days. Three positive clones were randomly selected for HIS3 and ADE2 detection. The diameters and colors of the colonies were observed and recorded.
4
Subcellular Localization of PmMYB7 Gene
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To further study the PmMYB7 gene, we used the bioinformatics analysis tool Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ , accessed on 30 November 2021) to predict the subcellular localization [59 (link)]. To verify the prediction, we used the pJIT166 vector to construct a recombinant plasmid containing a green fluorescent protein (GFP) marker and the PmMYB7 protein. The pJIT166 vector was digested at the HindIII and XbaI sites using QuickCut enzymes (Takara). Then, the PmMYB7 coding sequence (without stop codon) was inserted into the pJIT166 vector. The 2x35S::PmMYB7-GFP fusion vector was transformed into the E. coli strain TOP10 (Weidi). Positive E. coli cells were cultured, and high-quality plasmids were extracted. The plasmids were transformed into leaf protoplasts of A. thaliana by the PEG-mediated method [60 (link)]. After 14 h, we observed and obtained images of the GFP fluorescence signal by using a laser scanning confocal microscope (LSM710, Zeiss, Jena, Germany).
5
Construction of pGADT7-based cDNA Library
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The cDNAs of P. massoniana were ligated to the pGADT7 vector to construct recombinant vectors. pGADT7 was digested at the NdeI and XhoI sites using QuickCut enzymes (Takara). The double-stranded cDNAs from the previous step were ligated with the linearized pGADT7 by homologous recombination. The ligation system contained 7 µL of cDNA, 3 µL of enzymatically linearized library vector DNA, 5 µL of All-Direct recombination enzyme (Biogene, Shanghai, China) and 5 µL of ddH2O. These components were incubated at 50 °C for 1 h. The recombination reaction was inactivated by adding 2 µL of proteinase K (Sigma–Aldrich, St. Louis, MO, USA), followed by the addition of 1 µL of 20 µg/µL glycogen (Sigma–Aldrich), 50 µL of 7.5 M NH4Ac and 375 µL of anhydrous ethanol. The components were mixed, and the reaction mixture was stored at −80 °C for at least 1 h. The recombination product was collected and suspended in 10 µL of DEPC ddH2O on ice.
6
Overexpression of LtuBOP2 in Arabidopsis
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To overexpress the LtuBOP2 gene in A. thaliana, the LtuBOP2 coding regions were amplified and inserted at the XbaI and BamHI sites of the transformed pBI121 vector downstream of the 35S (Cauliflower mosaic virus) promoter using QuickCut enzymes (Takara, Dalian, China); further, the plasmid 35S::pBI121-LtuBOP2 was generated using the One Step Cloning Kit (ClonExpress® Ultra Vazyme Biotech Co. Ltd., Nanjing, China). We further imported the recombinant plasmids into Agrobacterium tumefaciens (GV3101) and transformed it into A. thaliana, applying the floral dipping approach [56 (link)]. Kanamycin-resistant primary transformants were gained on 1/2 MS agar plates. Taking the genomic DNA of transgenic leaves in A. thaliana as templates, a PCR reaction was accomplished to identify T1 transgenic plants by exploiting specific primers. We acquired stable genetic transgenic plants after the continuous screening of three generations.
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