Computational protocol: Structure, organization and evolution of ADP-ribosylation factors in rice and foxtail millet, and their expression in rice

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Protocol publication

[…] Primers were designed from 3′UTR of each OsARF/ARL transcript sequence using Primer3 v0.4 (http://bioinfo.ut.ee/primer3-0.4.0/) (). The total RNA was isolated using RNeasy plant mini kit (Qiagen, USA) following manufacturer’s instruction, and the quality and quantity was determined using 1.2% formamide denaturing agarose gel electrophoresis and ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). First strand cDNA synthesis was performed with 1 μg RNA using iScriptTM cDNA synthesis kit (Bio-Rad, Hercules, CA). Semi-quantitative and quantitative real-time PCR were performed in three biological replicates following the procedure described earlier, using OsAct1 gene as the internal control. The reaction mixtures, experimental conditions, melt curve analysis, and agarose gel electrophoresis were performed following Baisakh et al.. [...] Genomic DNA was isolated from all five rice cultivars following the miniCTAB protocol as described earlier. One hundred ng of DNA was amplified with gene-specific primers (http://bioinfo.ut.ee/primer3-0.4.0/) (). PCR reaction set up and thermal profile were same as described earlier. PCR products were cloned into pGEM T-easy vector (Promega, Madison, WI) following manufacturer’s instruction. Insert-containing plasmids were sequenced using an in-house ABI 3130 xL DNA analyzer (Applied Biosystems Inc., Carlsbad, CA). The sequences were filtered for removing vector sequence using VecScreen (http://www.ncbi.nlm.nih.gov/tools/vecscreen/) and aligned using BioEdit v7.2.5. […]

Pipeline specifications

Software tools Primer3, VecScreen, BioEdit
Applications Metagenomic sequencing analysis, qPCR
Organisms Oryza sativa, Setaria italica
Chemicals Abscisic Acid, Adenosine Diphosphate