The full-length HvMADS8 cDNA was amplified from inflorescence cDNA and cloned into the pGADT7 vector (Clontech) for in vitro translation using a TNT T7/SP6 Coupled Wheat Germ Extract System (Promega). The CArG-box and its flanking sequence in the promoter region of HvMADS13 and HvMADS21 were synthesized as FAM-labeled primers at Generay Biotech (China). 10× unlabeled probes were used as competitor. The probe preparation and assays were performed as described previously (Zhu et al. 2022 (link)). Primers are listed in Supplemental Data Set 5 .
Tnt t7 sp6 coupled wheat germ extract system
Manufactured by Promega
Sourced in Japan
The TNT T7/SP6 Coupled Wheat Germ Extract System is a cell-free protein expression system that allows for the in vitro synthesis of proteins from DNA templates. It utilizes the wheat germ extract as the source of cellular components necessary for transcription and translation, along with T7 or SP6 RNA polymerase to drive the expression process.
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2 protocols using tnt t7 sp6 coupled wheat germ extract system
1
HvMADS8 cDNA Cloning and Promoter Analysis
2
Transcription Factor Binding Assay
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The full-length cDNAs of OsMADS5 and OsMADS34 were cloned into the vector pGADT7 (Clontech, TaKaRa, Shiga, Japan) for in vitro transcription/translation (TNT T7/SP6 Coupled Wheat Germ Extract System; Promega). Fluorescein amidite (FAM)-labelled probes were generated by annealing two complementary primers containing FAM at the 5 0 -end. The binding reaction mixture contained 25 mM Tris-acetate (pH 7.5), 1 mM DTT, 0.1 mg ml À1 BSA, 2 mM MgAc, 20 nM FAM-labelled DNA, and 3 ll of in vitro synthesized protein. The binding reaction was performed for 30 min at 25°C before loading on a 6% native polyacrylamide gel. Competition was tested using 100-fold excess of nonlabelled probes. FAM-labelled probes were visualized using the FAM channel of a ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA). Primers are listed in Table S1.
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