The next Knowledge Exchange session I watched from the Nanopore Learning Lesson Library was “A primer on nucleic acid extraction.” Vania Costa spoke about protocols for extraction of DNA and RNA from samples and the available tested protocols. For DNA extraction, different kits produce different fragment sizes. Costa focuses on improving extraction for Nanopore sequencing. Costa noted that the requirements of the library prep and desired length of DNA will depend on the length and quality of nucleic acids that are extracted… and also the yield of this process. They used commercial rabbit blood and tested several kits. Costa also tested some RNA extraction methods. They recommended the Qubit fluorometer system for quantification of DNA. NanoDrop spectrophotometers can be used to identify possible contaminants. Assessment of fragment length can be done with a variety of methods. The Agilent BioAnalyzer or TapeStation and an ordinary agarose gel can be used. Sizing also helps with the molar quantification of the samples. Costa recommended storage at 4C for short term, and -20C for long term for blood. They also recommended reduced freeze thaw cycles because the N50 decreases. Interestingly, they mentioned that storage of DNA at 4C is “ok” up to six months! Costa explained that several protocols are available in the Nanopore Community forum. I will try to follow this forum to learn about improvements in extraction and suggestions.
