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Benjamin Schwessinger and Megan McDonald 2017. High quality DNA from Fungi for long read sequencing e.g. PacBio, Nanopore MinION. protocols.io dx.doi.org/10.17504/protocols.io.k6qczdwCopy Citation Copied
URL: https://dx.doi.org/10.17504/protocols.io.k6qczdw
Authors: Benjamin Schwessinger and Megan McDonald
Group: Solomon Lab - Australian National University, High molecular weight DNA extraction from all kingdoms, Zymoseptoria community protocols (STBnet)
Summary: Extraction of high quality DNA for long read sequencing e.g. PacBio and MinIonOptimized for DNA extraction from Bipolaris sorokiniana. Also tested on Parastaognospora nodorum, Zymoseptoria tritici, wheat stripe rust, barley stripe rust and Pyrenophora tritici-repentisBuffers 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.
Affiliations: Australian National University
Version: 4
Publication Date: 2017
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Source: Protocols.io
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