Genotyping Arabidopsis T-DNA lines v1

Steven J Burgess


This protocol is used for genotyping Arabidopsis seedlings to test for the presence of a transfer DNA (T-DNA) insertion. By using two primer sets it is possible to determine whether a seedling is homozygous, heterozygous or azygous for an insertion in the predicted genomic location. To identify lines with T-DNA insertions in a gene of interest, you need the Arabidopsis Genome Identifier (AGI) number corresponding to the genomic locus (e.g. RCS1A = AT1G67090), then visit the Salk Institute T-DNA Express site to find all the mapped insertions at your locus of interest. Genotyping primers have been pre-designed for each T-DNA line, these can be retrieved from the Salk Institute T-DNA primer site, and ordered at any supplier of DNA oligonucleotides before starting the protocol. In the US T-DNA lines can be purchased from the Arabidopsis Biological Resource Center (ABRC) and in the UK and EU from the European Arabidopsis Stock Center (NASC). Recommended reading Setting up the PCR reaction Genotyping is performed with the Phire Direct PCR Mix, this includes the polymerase, nucleotides and salts necessary for amplification. We use the “dilution protocol” which involves taking a small leaf disk...Continue Reading

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