Background: High-molecular-weight and pure DNA is crucial for high-quality results from 3rd generation DNA Analyzers and optical mapping technologies. Conventional nuclei isolation methods for preparing high-molecular-weight genomic DNA from plant tissues include the preparation of protoplasts or embedding nuclei in an agarose matrix with subsequent manipulations via electro-elution or pulsed-field gel electrophoresis.
Results: In this method, plant nuclei are isolated by physically grinding tissues and reconstituting the intact nuclei in a unique Nuclear Isolation Buffer (NIB). The plastid DNAs are released from organelles and eliminated with an osmotic buffer by washing and centrifugation. The purified nuclei are then lysed and further cleaned by organic extraction, and the genomic DNA is precipitated with a high concentration of CTAB. The highly pure, high molecular weight gDNA is extracted from the nuclei, dissolved in a high pH buffer, allowing for stable long-term storage.
Conclusions: This method is unique and avoids the use of embedding in agarose, which dramatically reduces time (4-8 h versus days), complexity, and materials cost. This procedure can be used on essentially any plant species and tissue stage. Here we describe a case study and a simple method to rapidly prepare high molecular weight gDNA from Upland cotton, blackgrass, and strawberry suitable for single-molecule sequencing.
© The Author(s) 2020.