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Wizard Genomic Dna Purification Kit Protocol

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40 views

Wizard Genomic Dna Purification Kit Protocol

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MB avonpclk.com
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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3.E.

Isolating Genomic DNA from Plant Tissue

Materials to Be Supplied by the User


• 1.5ml microcentrifuge tubes
• liquid nitrogen
• microcentrifuge tube pestle or mortar and pestle
• water bath, 65°C
• water bath, 37°C
• isopropanol, room temperature
• 70% ethanol, room temperature
1. Process leaf tissue by freezing with liquid nitrogen and grinding into a fine powder using a microcentrifuge tube
pestle or a mortar and pestle. Add 40mg of this leaf powder to a 1.5ml microcentrifuge tube.
2. Add 600µl of Nuclei Lysis Solution, and vortex 1–3 seconds to wet the tissue.
3. Incubate at 65°C for 15 minutes.
4. Add 3µl of RNase Solution to the cell lysate, and mix the sample by inverting the tube 2–5 times. Incubate the
mixture at 37°C for 15 minutes. Allow the sample to cool to room temperature for 5 minutes before proceeding.
5. Add 200µl of Protein Precipitation Solution, and vortex vigorously at high speed for 20 seconds.
6. Centrifuge for 3 minutes at 13,000–16,000 × g. The precipitated proteins will form a tight pellet.
7. Carefully remove the supernatant containing the DNA (leaving the protein pellet behind) and transfer it to a
clean 1.5ml microcentrifuge tube containing 600µl of room temperature isopropanol.
Note: Some supernatant may remain in the original tube containing the protein pellet. Leave this residual liquid
in the tube to avoid contaminating the DNA solution with the precipitated protein.
8. Gently mix the solution by inversion until thread-like strands of DNA form a visible mass.
9. Centrifuge at 13,000–16,000 × g for 1 minute at room temperature.
10. Carefully decant the supernatant. Add 600µl of room temperature 70% ethanol and gently invert the tube several
times to wash the DNA. Centrifuge at 13,000–16,000 × g for 1 minute at room temperature.
11. Carefully aspirate the ethanol using either a drawn Pasteur pipette or a sequencing pipette tip. The DNA pellet is
very loose at this point and care must be used to avoid aspirating the pellet into the pipette.
12. Invert the tube onto clean absorbent paper and air-dry the pellet for 15 minutes.
13. Add 100µl of DNA Rehydration Solution and rehydrate the DNA by incubating at 65°C for 1 hour. Periodically
mix the solution by gently tapping the tube. Alternatively, rehydrate the DNA by incubating the solution over-
night at room temperature or at 4°C.
14. Store the DNA at 2–8°C.

12 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516
TM050 · Revised 3/19 www.promega.com

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