The E.Z.N.A.^® High Performance (HP) Plant DNA Kit is designed for efficient recovery of genomic DNA up to 60 kb in size from fresh and dried plant tissue samples rich in polysaccharides or those with lower DNA contents. Up to 100 mg of wet tissue (or 30 mg dry tissue) can be processed in less than 30 minutes.

Principle

The E.Z.N.A.^® High Performance (HP) Plant DNA Kit combines the reversible nucleic acid-binding properties of the HiBind^® matrix with the speed and versatility of spin column technology to eliminate polysaccharides, phenolic compounds, and enzyme inhibitors from plant tissue lysates. This procedure relies on the well established properties of the cationic detergent, cetyltrimethyl ammonium bromide (CTAB), in conjunction with the selective DNA binding HiBind^® matrix. Samples are homogenized and lysed in a high salt buffer containing CTAB and extracted with chloroform to remove polysaccharides and other components that interfere with many routine DNA isolations and downstream applications. Following a rapid alcohol precipitation step, binding conditions are adjusted and DNA is further purified using HiBind ^® DNA spin columns. The use of mini-columns facilitate the binding, washing, and elution steps thus enabling multiple samples to be simultaneously processed. For more information on columns, click Flexible Multi-Format Columns.

Procedure

To 10-50 mg powdered dry tissue add Buffer CPL, mix it well and incubate at 65°C for 30 minutes Add chloroform/isoamyl alcohol (24:1), vortex to mix and centrifuge to pellet the cell debris and other contaminants. Add 0.7 volume isopropanol to the supernatant and vortex to precipitate DNA. Immediately centrifuge at 10,000 x g for 5 min to pellet DNA. Discard the supernatant and re-suspend the DNA pellet in sterile de-ionized water pre-heated to 65°C. After a brief incubation, add RNase and mix. Adjust binding conditions of the sample by adding Buffer CAD followed by absolute ethanol and vortex to obtain a homogeneous mixture. Apply the entire sample to a HiBind^® DNA column and centrifuge to bind DNA. Wash the column twice with DNA wash buffer and centrifuge the empty column for 2 min at maximum speed to dry. Elute the DNA with Tris buffer pH 9.0 or 8.5 (or, sterile de-ionized water) pre-warmed to 65°C.

Applications

DNA purified using the E.Z.N.A.^® High Performance (HP) Plant DNA Kit is suitable for the following applications :

• PCR
• Restriction Digestion
• Hybridization Techniques