Generacer race ready cdna kit

Total RNA from two 2 cm diameter leaf discs was isolated using the Trizol Reagent (Sigma-Aldrich) method following the manufacturer’s instructions. First-strand cDNAs were synthesized from 2 ug of total RNA using Superscript II RNAse H_ Reverse Transcriptase (Invitrogen). cDNAs for tobacco and Nicotiana benthamiana POB1 were amplified by PCR using gene- specific primers (S1 Table). RACE experiments were performed using a GeneRacer RACE Ready cDNA Kit (Invitrogen, USA) and according to the manufacturer’s instructions but with gene-specific primers.

The full-length cDNA of the putative NbFIE genes were amplified using the primers NbFIEfF/NbFIEfR. One microlitre of 20 μl first-strand cDNA reaction product was used as a template for PCR. The PCR products were gel purified and cloned with a pEASY-T1 cloning kit (TransGen Biotech, Beijing, China), and then sequenced to verify their identity as two putative homologues of the FIE gene. The sequence alignment was done using Clustal W (http://srs.ebi.ac.uk/) while the phylogenetic tree was constructed using Bioedit software. To clone the NbGH3.6 full-length cDNA, the 3′-RACE and 5′-RACE reaction was performed with the GeneRacer RACE Ready cDNA kit (Invitrogen, USA). The cDNA from the NbFIE-silenced plants acted as the PCR template and the NbGH3.6 gene-specific primers are listed in Supplementary Table S3 at JXB online.

The reactions of Rapid Amplification of cDNA Ends (RACE) were performed using GeneRacer™ RACE Ready cDNA Kit (Invitrogen) strictly following the manufacturer's instructions. The amplified fragments were then transferred into the pMD18-T vector (Takara Bio), nucleotide sequences were determined with CEQ 2000XLDNA Analyzer (Beckman Coulter), and the data were analyzed and visualized by using Sequencer (Genes Codes Corporation). Three independent clones of each amplification product were sequenced to avoid errors caused by PCR. The primers used for this experiment can be found in Table 1.