MasterPure™ Plant Leaf DNA Purification Kit

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Last modified: 2008-07-15 14:57:15

The MasterPure™ Plant Leaf DNA Purification Kit is designed for isolating DNA from 35-100mg of plant leaf tissue. The protocol can be completed in less than one hour and may be scaled up to obtain larger amounts of DNA. The purified DNA can be used for PCR amplification, restriction digestion, and Southern blotting.

The simple purification procedure involves incubating ground plant leaf tissue in Plant DNA Extraction Solution for 30 minutes at 70°C. The sample is then chilled, subjected to centrifugation, and the DNA is precipitated from the supernatant with isopropanol. Following resuspension in Cleanup Solution, the DNA is precipitated again with isopropanol. The kit was optimised with the grapevine, Vitis vinifera, whose leaves are notoriously high in polyphenolic (tannin-like) compounds and polysaccharides, which interfere in both the isolation and subsequent utilisation of DNA. No enzymatic digestions, CTAB (hexadecyltrimethylammonium bromide), or organic solvents are used in the MasterPure protocol.

Last modified: 2008-05-14 16:46:46

Applications

• Identification of plant species by microsatellite typing (figure 1)
• PCR amplification, restriction digestion, and Southern blotting

Benefits

• Versatile - recover DNA from leaves high in polysaccharides and polyphenolic compounds
• Rapid - purify DNA in less than one hour
• Simple and non-toxic
• Recover DNA of higher integrity than column-based purification methods

Figure 1. Higher yields of DNA isolated with the MasterPure™ Kit versus another supplier's kit. 10 µl of each DNA preparation (either MasterPure or Supplier G) were separated by electrophoresis in a 0.7% agarose gel, the gel was stained with SYBR® Gold, and viewed with a Dark Reader™ Transilluminator. Lanes M, 1-kb DNA ladder; Lanes 1-4, Pinot noir grape leaf DNA purified with the MasterPure Kit; Lanes 5-8, Pinot noir grape leaf DNA purified with Supplier G Kit.

MasterPure Plant Leaf DNA Purification Kit Protocol

1. Grind 35-100 mg of leaf tissue in a 1.5-ml microcentrifuge tube containing Plant DNA Extraction Solution.
2. Heat for 30 minutes at 70°C.
3. Chill on ice and centrifuge 5 minutes to pellet cellular debris. Transfer supernatant to a fresh tube and repeat the centrifugation step. Transfer supernatant to a fresh tube.
4. Precipitate DNA with isopropanol and resuspend pellet in Cleanup Solution. Then, precipitate DNA again with isopropanol, wash pellet with 70% ethanol, and resuspend DNA in TE Buffer.
5. If the DNA solution has detectable color, repeat Step 4.

Figure 2. Restriction digests of DNA isolated with the MasterPure™ Kit versus a column-based kit. Lane 2 shows tobacco DNA purified with the MasterPure Plant Leaf DNA Purification Kit, digested with Ssp I, a "finicky" restriction enzyme. Lane 3 shows the same digestion using DNA purified with Supplier Q's column-based kit. DNA purified with the MasterPure Kit was cleaved by Ssp I, as seen by the existence of a repetitive element band. The repetitive element is not cleaved by Ssp I using DNA purified with the Supplier Q Kit.

Last modified: 2008-05-14 16:46:46

Protocols for: MasterPure™ Plant leaf DNA Purification Kit

(catalogue number MPP92010 / MPP92100)

Please note: all protocols off site are the responsibility of the products supplier

Last modified: 2008-05-14 16:46:46