Triticum aestivum CAP12
Triticum aestivum
Plants were grown in greenhouse conditions under natural day light supplemented with artificial source of light. The total RNA was isolated from 4 week seedlings using the RNeasy Plant Mini Kit (QIAGEN). First-Strand cDNA synthesis was performed according SMART cDNA synthesis technology (Clontech Laboratories, Inc.) using 3' SMART CDS modified Primer II A primer (5'-AAG CAG TGG TAT CAA CGC AGA GTA CTT TTG T(9) C T(10) VN-3') and SuperScript III reverse transcriptase (Invitrogen). Double-stranded cDNA was amplified by long-distance (LD) PCR using Advantage 2 PCR Enzyme System (Clontech Laboratories, Inc). Amplification was performed on thermal cycler (Applied Biosystem) with the following PCR parameters: 95oC for 1min; 95oC-15sec, 65oC-30sec, 68oC-6min - 16 cycles; 4oC- 45 min (optional). Double-stranded cDNA was checked on the 1.1% agarose/EtBr gel in 1XTAE buffer and purified with QIAquick PCR Purification Kit (QIAGEN). The double-stranded cDNA was normalized using TRIMMER cDNA normalization Kit (EVROGEN), which is based on a unique DSN (duplex-specific nuclease) normalization technology and is specially developed for normalization of cDNA enriched with full-length sequences.