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URL: https://dx.doi.org/DOI:10.17504/protocols.io.ewtbfen

Authors: Benjamin Schwessinger

Group: High molecular weight DNA extraction from all kingdoms, RUSTIS

Summary: Extraction of high quality DNA for long read sequencing e.g. PacBioOptimized for DNA extraction from wheat stripe rust spores and also tested on barley leaf rust.Buffers are best when fresh and not older than 3-6 months. Buffered Phenol:Chloroform:Isoamylalcohol (25:24:1) should not be older than 3 months.Critical steps to obtain high quality DNA:Do NOT heat samples during DNA extractions! Perform all steps at RT or 4oC as indicated.Do NOT incubate samples with KAc for prolonged time periodsPerform two steps of buffered Phenol:Chloroform:Isoamylalcohol purification to reduce co-purifying metabolites.DNA fragments were well above the 40kb mark based on Pippin Pulse Gels. The sequencing center performed a second AMPure purification step before library construction. Summary statistics of sequencing runs to follow.

Associated Publications: Langner T, Harant A, Gomez-Luciano LB, Shrestha RK, Malmgren A, Latorre SM, Burbano HA, Win J, Kamoun S, Genomic rearrangements generate hypervariable mini-chromosomes in host-specific isolates of the blast fungus. PLoS Genetics 17(2). doi: 10.1371/journal.pgen.1009386

Affiliations: ANU

External URL: https://doi.org/10.1371/journal.pone.0238611

Version: 10

Publication Date: 2016

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