High quality DNA from Fungi for long read sequencing e.g. PacBio v8 [dataset]

Benjamin Schwessinger
2016 protocols.io  
1 Extraction of high quality DNA for long read sequencing e.g. PacBio Optimized 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
more » ... prolonged time periods Perform 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.
doi:10.17504/protocols.io.ewdbfa6 fatcat:5rurkkiuzvbdjbxnetcpaw2lqe