Stella Loke from Deakin University in Australia spoke at London Calling 2019 about “Optimising plant DNA extraction for nanopore sequencing.” We are considering sequencing plant DNA this summer and, therefore, want to learn more about plant DNA challenges. Loke spoke about wanting the nano spec readings from Qubit to match the nano spectrophotometer. Loke confirms DNA concentrations with a TapeStation. Plants often have waxy coatings and carbohydrates that affect sequencing with Nanopore devices. Loke noted that tissue choice is important: leaves are better than stems, and actively dividing cells are desired. Loke noted that washing the ground tissue in cold Tris-HCl pH 8.0 with 200 nM EDTA. Loke cuts tissues and uses liquid nitrogen. Loke recommends SDS or CTAB for DNA extraction. The ground powder is heated in a denaturant several times before being extracted with phenol chloroform. CTAB modifications and BME are included. SDS extraction is the second method. Phenol:chloroform: isoamyl alcohol extractions are needed. Loke also uses commercial kits, including DNeasy Plant kits. Ampure beads don’t clean up things bound to DNA. Loke also uses the QIAGEN PowerClean to remove inhibitors and polysaccharides. I didn’t know about this kit, but Loke highly recommended it. Loke pools a couple of extractions to obtain the DNA yield. This session provided several useful tips!
