Background
High-molecular-weight and pure DNA is crucial for high-quality
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.
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.