Superscript 2 reverse transcriptase

P. putida KT2440 and its derivatives were cultivated in MSM supplemented with the appropriate carbon sources. Total bacterial RNA was purified by the use of an RNAprep Pure cell/bacteria kit (Tiangen Biotech, China) according to the manufacturer’s directions. Contaminating DNA in the RNA preparations was removed by the use of RNase-free DNase I (TransGen, China). Synthesis of cDNA was performed using Superscript II reverse transcriptase (TransGen, China).
For transcriptional and cotranscriptional analysis, RT-PCR analyses were performed using mRNAs of P. putida KT2440 cells cultured in MSM and the appropriate primers. RT-qPCR was performed by the use of TransStart Top Green qPCR SuperMix (TransGen, China) and a LightCycler 480 system (Roche). The relative levels of expression of the genes were calculated using the threshold cycle (2^−ΔΔCT) method (50 (link)). The results were normalized to the 16S rRNA gene level.

Total RNA was extracted using Trizol (Tiangen, Beijing, China) from the trifoliolate leaves of ZGDD seedlings sampled on day 9 after commencing LD treatment; cDNA was synthesized with Superscript II reverse transcriptase (TransGen Biotech, Beijing, China) and used as a template for further GmFT1a cDNA cloning. Amplification was performed using PCR reactions with a KOD‐plus‐Neo DNA polymerase kit (Toyobo, Tokyo, Japan).