qRT-PCR of the resulting cDNA was performed with gene-specific primers (Table 1) on a CFX Connect Real-Time PCR Detection System (Bio-Rad, USA) with a SYBR Premix Ex Taq (Tli RNaseH Plus) Kit (TaKaRa, Japan). One microgram of qualified total RNA was subjected to reverse transcription with a PrimeScript RT reagent Kit with gDNA Eraser per the manufacturer’s instructions (TaKaRa, Japan).
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Quantitative Real-time PCR.Īfter harvesting bacteria from LB medium, purification of total RNA was performed using RNAiso Plus reagent (TaKaRa, Japan) following the protocol described by the manufacturer. Antibiotics were supplemented as required at the following concentrations: 100 μg/mL of ampicillin, and 20 μg/mL of gentamycin. KPM minimal medium was adopted for all nitrogen fixation assays and contained per liter 1040 mg Na 2mg KH 2PO 4, 26 mg CaCl 2♲H 2O, 30 mg MgSO 4, 7.5 mg Na 2MoO 4♲H 2O, 0.3mg Mmg glucose, 500 mg casein hydrolysate, 36 mg ferric citrate, 10 mg para-aminobenzoic acid, 5 mg biotin, and 1 mg vitamin B 1, supplied with 10 mM (NH 4) 2SO 4 (KPM-HN) for pregrowth or 10 mM glutamate (KPM-LN) for nitrogenase activity assays. coli JM109 growth contained 10g/L tryptone, 10 g/L NaCl, and 5 g/L yeast extract. EJN transformed with pJQ200SK- OmpA/PbrR was selected from LB agar plates containing appropriate antibiotics, and the resulting strain was designated EJNC. After confirmation by sequencing, the PCR product was digested with Kpn I and Hind III and then insert into pJQ200SK to yield pJQ200SK- OmpA/PbrR.
For construction of the second plasmid, pJQ200SK OmpA/PbrR (with a compatible p15A ori), a lab store plasmid pBAD24- OmpA/PbrR was used as the template to PCR-amplify OmpA/PbrR with P200F and P200R primers. coli JM109, and the resulting recombinant was designated EJN. A high-copy plasmid, pUC57- nif (pMB1 ori), harboring the minimal nitrogen fixation gene cluster ( nif) of Paenibacillus polymyxa CR1 was chemically synthesized and then transformed into E. Escherichia coliJM109 was purchased from Takara and designated EJ. The bacterial strains, plasmids and primers used in this study are all listed in Table 1.
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