Computational protocol: Diversity of Stability, Localization, Interaction and Control of Downstream Gene Activity in the Maize Aux/IAA Protein Family

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Protocol publication

[…] Total RNA was extracted with the RNeasy Mini Kit (Qiagen, http://www.qiagen.com/) from 100 mg frozen material of five primary roots per biological replicate, followed by RNase-free DNAse I treatment (Fermentas, http://www.thermoscientificbio.com/fermentas/). For cDNA synthesis 1 µg of total RNA was amplified using the Revert Aid H Minus First Strand cDNA Synthesis Kit (Fermentas). To clone the open reading frames of ZmIAA2 (GRMZM2G159285), ZmIAA11 (GRMZM2G059544), ZmIAA15 (GRMZM2G128421), ZmIAA20 (GRMZM5G864847) and ZmIAA33 (GRMZM2G359924) oligonucleotide primers were designed by PrimerPlus3 software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) and checked with NetPrimer Software (PREMIER Biosoft, http://www.premierbiosoft.com/). PCR amplicons of the Aux/IAA open reading frames generated by specific oligonucleotide primers were introduced into the vector pGEM-t-easy (Promega, http://www.promega.de/). Subsequently, the conserved degron-Sequence VGWPPV in domain II was mutated in all Aux/IAA genes via the GENEART site-directed mutagenesis system (Life technologies, http://www.lifetechnologies.com/). Either the first proline residue was substituted by serine (VGWSPV) or the second proline was replaced by leucine (VGWPLV). The oligonucleotides were designed according to the manufacturer's suggestions (). […]

Pipeline specifications

Software tools Primer3, NetPrimer
Application qPCR
Organisms Zea mays