The sources of the strains and plasmids used in this work are listed by their associated method in Table 2 . The custom DNA oligonucleotides and primers (Sigma) used in this study are listed in Table S2 in the supplemental material. EmeraldAmp GT master mix (Clontech) was used for all PCRs unless explicitly stated. ZymoPURE midiprep, DNA Clean & Concentrator 5 (DNA C&C), and Oligo Clean & Concentrator (Oligo C&C) kits were purchased from Zymo Research. Restriction enzymes, ligases, and RecA were purchased from NEB. Sequencing was performed by the Oklahoma Medical Research Foundation DNA Sequencing Core facility.
Ligases
Manufactured by New England Biolabs
Ligases are enzymes that catalyze the formation of covalent bonds between two molecules. They are commonly used in molecular biology and genetic engineering to join DNA fragments together, facilitating the construction of recombinant DNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Restriction enzyme, by New England Biolabs (3 mentions)
Onetaq quick load 2x master mix, by New England Biolabs (1 mentions)
13c methanol, by Cambridge Isotopes (1 mentions)
Gibson assembly master mix kit, by New England Biolabs (1 mentions)
Phusion dna polymerase, by New England Biolabs (1 mentions)
Microtof spectrometer, by Bruker (1 mentions)
Ultraflex 3 maldi tof mass spectrometer, by Bruker (1 mentions)
Hek293 freestyle cells, by Thermo Fisher Scientific (1 mentions)
Site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Pcdf 1b, by Merck Group (1 mentions)
Geneart, by Thermo Fisher Scientific (1 mentions)
5 protocols using ligases
1
Bacterial Strain and Plasmid Sources
2
Isotope Labeling and Metabolite Analysis
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13C methanol of 99 % purity was purchased from Cambridge Isotope Laboratories (Tewksbury, MA). All other chemicals including metabolite standards were purchased from Sigma-Aldrich (St. Louis, MO). Phusion DNA polymerase, dNTP, buffer, ligases, OneTaq Quick-Load 2X Master Mix and the Gibson Assembly Master Mix kits used in this study were from New England Biolabs (Ipswich, MA). Primers, the sequences of which are shown in Table 1 , were obtained from Invitrogen (Grand Island, NY) and IDT (Coralville, IA). Acetonitrile and water used as UPLC solvents were UPLC-MS grade. Hypho minimal medium as previously described [24 (link)] was used for 13C flux analysis cell culture and growth rate determination, involving two different concentrations of cobalt (1.35 and 6.31 μM respectively).
3
Characterization of Synthetic Compounds
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All reagents were purchased from Sigma Aldrich, Thermo Fisher, Alfa Aesar, Apollo Scientific, or Fluorochem and used without further purification. Restriction enzymes, polymerases, and ligases were purchased from New England Biolabs. MALDI-TOF MS was carried out using ground steel target plates on a Bruker ultraFlex III MALDI-TOF mass spectrometer. NMR spectra were obtained using a Bruker 400 MHz NMR spectrometer (Bruker AV3400HD). ESI-MS data were obtained on a Bruker MicroTOF spectrometer.
4
Cloning and Protein Expression Protocols
5
Overexpression of GA Biosynthesis Genes in Plants
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The whole coding sequences of PpGA20ox1, − 2, − 5, PpGA3ox1 and PpGA2ox6 were amplified using cDNA synthesized from shoot tips of ‘QMH’ as templates. The PCR product and pSAK277 vector were digested with a restriction enzyme, and then joined together using ligases (NEB, Beijing). Arabidopsis transformation was performed according to the floral dip method [44 (link)]. For transgenic plant selection, T0 seeds were sterilized and germinated on Murashige and Skoog (MS) medium containing 12 μg mL− 1 kanamycin. Kanamycin-resistant plants were transplanted to soil and placed in a growth chamber at 25 °C and 65– 80% relative humidity. Phenotype analysis was conducted in the T3 generation.
Tobacco (Nicotiana tabacum) was transformed using the leaf-disc method [45 (link)]. More than five transgenic plants were selected using Murashige and Skoog (MS) medium containing 50 μg mL− 1 kanamycin and were confirmed by PCR using specific primers for the PpGA2ox6 gene.
Tobacco (Nicotiana tabacum) was transformed using the leaf-disc method [45 (link)]. More than five transgenic plants were selected using Murashige and Skoog (MS) medium containing 50 μg mL
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