Machery-Nagel NucleoSpin® 96 Plant Genomic DNA - Xiril - #1

Text version of the page

Fully Automated Purification of Genomic DNA from Plant Using NucleoSpin® 96 Plant on the Xiril X100 Platform with Vacuum Filtration High-throughput extraction of high molecular-weight genomic DNA is the first and often limiting step when processing large numbers of different plant samples. In the fields of plant research and crop design, high reproducibility in yield and quality is mandatory for an optimal performance of downstream processes like genotyping or PCR-based applications e.g. SNP analysis. MiniNote 14/2007-NS 96 Plant on Xiril X100 The automated extraction system described here meets all requirements for a robust and user-friendly procedure. Manual interactions are minimized due to automated liquid handling, robotic plate handling, integrated shaking, heating, and vacuum filtration. Optionally, initial sample homogenization can also be automated. With the NucleoSpin® 96 Plant kit excellent DNA yields and outstanding quality of DNA from plant material is achieved. The extracted DNA is suitable for common downstream applications like restriction analysis, Southern Blotting, or PCR. Features: • Fully automated (after homogenization of plant material). • Flexibility: gDNA from up to 80 mg plant material. • Fast: 100 minutes for 96 samples (not including lysis). • Silica-membrane technology. • Elution volume: 75 – 200 ìl. • High yields: up to 30 ìg DNA from plant tissue. • High consistency: typical CVs for yield are <15%. • High DNA quality: fragment size up to 30 – 50 kbp, purity A260/280 1.8 – 2.0. • Efficient removal of PCR inhibitors. Instrumentation: The automation system consists of a Xiril X100 Robotic Workstation equipped with a plate gripper tool, a thermo shaker and solid phase extraction (SPE) unit for vacuum filtration (Fig. 1). The MACHEREY-NAGEL NucleoSpin® 96 Plant kit was used for high yield and high quality DNA extraction. Sample homogenization was performed using the Dispomix homogenization instrument (stand-alone device or integrated on Xiril platform). Alternatively, homogenization can be performed using Genogrinder or Mixer Mill. Fig. 1: Xiril X100 Automated Liquid Handling System equipped with gripper tool and Solid Phase extraction unit. Fig. 2: NucleoSpin® 96 Plant procedure with optional lysis clearing plate. For details on the procedure see below. Method: This application note describes the procedure using the Dispomix homogenization instrument and the use of a lysate clearing plate (see Fig. 2). Samples of lyophilized wheat leaves (corresponding to approx. 80 mg wet weight plant material) were homogenized using the Dispomix in lysis buffer C1 supplemented with RNase. For heat incubation 400 ìl of each sample were transferred to a square-well block and incubated with shaking for 30-60 minutes at 56°C. Crude lysates (350 ìl) were transferred to a lysate clearing plate, overlayed with 200 ìl ethanol and filtered into a square-well block. Binding buffer C4 (300 ìl) was added to the cleared lysate. After mixing and transferring to the NucleoSpin® Plant Binding Plate the genomic DNA is bound to the silica membrane upon vacuum filtration. After washing with high-salt buffer CW (600 ìl) and ethanolic buffer C5 (2x900 ìl) the silica membrane of the NucleoSpin® Plant Binding Plate is dried under vacuum for 10 minutes. Highly pure gDNA is then eluted in two steps (100 ìl each step) with Elution Buffer CE, prewarmed to 70°C to increase the yield of genomic DNA. The complete procedure is fully automated and the DNA is ready-to-use for common downstream Homogenize plant leaves using the Dispomix instrument Incubate the homogenate and transfer to Lysate Clearing Plate Adjust binding conditions, transfer into NucleoSpin® Binding Plate Bind, wash and elute highly pure gDNA MACHEREY-NAGEL