DNA was isolated from arabidopsis leaves using either CTAB lysis (CTAB) or the DNeasy Plant Maxi Kit (DNeasy). Amplification reactions were prepared using purified DNA (1: 50 pg; 2: 100 pg) and primers to the Akin 10 gene. M: markers. (Data kindly provided by Alain Lecharny, Institut de Biotechnologie des Plantes, UMR CNRS-UPS Orsay, France.)
DNA (10 ng) from the indicated leaves or needles was amplified using universal primers for the noncoding intergenic spacer between the tRNA genes trnL (UAA) 5' exon and trnL (UAA) 3' exon of cpDNA (Taberlet, P. et al. (1991) Plant Mol. Biol. 17, 1105). M: 100 bp ladder.
DNA (50 ng) from leaves of the indicated in vitro-propagated sunflower (Helianthus) species was amplified using a 10-base RAPD primer and separated on a 1.5% agarose gel. M: 100 bp ladder. (Data kindly provided by H. J. Henn, Institute of Agricultural Botany, University of Bonn, Germany.)
The DNeasy Plant Maxi Kit allows rapid and efficient isolation of high-quality DNA from a wide variety of plant species and tissue types including the most demanding sources (see table "Selection of plant species processed with DNeasy Kits"). Samples may be fresh, frozen, or dried. The optimized DNeasy Plant procedure incorporates the QIAshredder Maxi spin column, a unique filtration and homogenization column that efficiently removes cell debris and improves sample handling following lysis.
Young leaves or needles (and other tissues, as indicated) were collected and immediately flash frozen. DNA isolation was then performed with the DNeasy Plant Mini Kit. 1Beechnut, 2dried leaves, 3callus, 4leaves from adult plant, 5endosperm, 6old leaves, rich in carbohydrates, 7buds. For more information on DNA isolation from other species including fungi, call QIAGEN Technical Services or your local distributor.
The typical yield is 30–260 µg, with a sample size of up to 1 g wet weight, and an elution volume of 500 µl to 2 ml. The DNA yields vary between different species and tissues depending on genome size, ploidy, cell number, and age of tissue sample. Lower and higher range values correspond to arabidopsis and wheat, respectively. Absorbance scans of DNeasy purified DNA show a symmetrical peak at 260 nm (see figures "DNA purity from oak leaves and pine needles"), confirming that the DNA is free of impurities, including enzyme inhibitors. Purified DNA can be used in a wide range of applications (see figures "PCR performance", "PCR analysis", and "RAPD analysis").
DNeasy Plant Kits use advanced silica-membrane technology and simple spin procedures to isolate highly pure total cellular DNA from plant tissues and cells or fungi. DNeasy technology replaces cumbersome DNA isolation procedures such as cetyltrimethylammonium bromide (CTAB), phenol, or chloroform extraction. Using the DNeasy procedure, alcohol precipitation is not necessary — purified DNA is ready for immediate use. DNeasy sample preparation technology is fully licensed.
Samples are first mechanically disrupted and then chemically lysed (see flowchart "DNeasy Plant and DNeasy 96 Plant procedures"). RNA is removed by RNAse digestion during lysis. Cell debries, precipitated proteins, and polysaccharides are removed and the sample is homogenized by centrifugation through a QIAshredder Maxi spin column. Buffering conditions are adjusted and the lysate is loaded onto the DNeasy Plant Maxi spin column. During a brief spin, DNA selectively binds to the silica membrane while contaminants pass through. Remaining contaminants and enzyme inhibitors are removed in one or two efficient wash steps. Pure DNA is then eluted in water or low-salt buffer, ready for use.
The DNeasy Plant Maxi Kit provides purification of DNA from plant tissue, including:
PCR, real-time PCR, blotting
500 µl – 2 ml
Main sample type
Purification of total RNA, miRNA, poly A+ mRNA, DNA or protein
2.5 ml (100 mg/ml; 7000 units/ml, solution)
Unit definition: That amount of enzyme causing the hydrolysis of RNA at a rate such that k (velocity constant) equals unity (Kunitz units) at 25°C and pH 5.0.