MONOCOT DNA ISOLATION
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1465
- Collect fresh tissue and store at -20°C.
- Grind leaf tissue to a fine powder with liquid nitrogen in a prechilled mortar [Store mortar and pestle at -20°C or -80°C].
- Transfer ground tissue with a chilled paintbrush into a well-chilled, 50-ml polypropylene tube and store at -20°C. Do not let the powder thaw. All tools should be chilled in liquid nitrogen.
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Add sodium bisulfite to the extraction buffer and adjust pH with 5N NaOH to 7.8�8.0. Heat the extraction buffer to 65°C and add 15�20 ml to 10�15 ml of frozen tissue. Mix well.
DNA extraction buffer for 1 L [Final] 5M NaCl 100.0 mL500 mM1M Tris-HCl pH 8.0 100.0 mL100 mM0.5M EDTA pH 8.0 100.0 mL50 mM20% SDS 62.5 mL0.84% (w/v)Bring to final volume with sterile distilled water. Note: Add 0.38 g sodium bisulfite/100 mL extraction buffer just before use and readjust pH to 7.8�8.0 with 5N NaOH. - Incubate in a 65°C water bath for 30 min; invert the tubes every 5�10 minutes.
- Add chloroform:isoamyl alcohol [24:1 v/v] to the top of the tube and mix vigorously.
- Centrifuge 15 min at 4,500 rpm. Transfer upper phase into a new 50-ml tube by pouring through 2�4 layers of cheesecloth or pipette off the upper phase if the interphase does not appear solid.
- Add 2 volumes (fill tube to top) of cold [-20°C] 95% EtOH and mix gently to precipitate the DNA. Place at -20°C for 30�60 min.
- Pour off the 95% EtOH, wash with fresh 70% EtOH, and add 30 ml of cold 70% EtOH. Mix gently for a few minutes. DNA can be left indefinitely in 70% EtOH [can leave overnight at -20°C].
- Rewash with fresh 70% EtOH if the DNA is still discolored. Remove DNA by hooking it on a Pasteur pipette and blot off excess 70% EtOH with a Kimwipe. Transfer DNA to the bottom of a sterile 1.5-ml microfuge tube. If making many extractions, centrifuge wet DNA gently, then invert tube a few minutes to let the liquid drain out. Dry DNA at RT for 2�3 hours.
- Dissolve DNA in sterile TE [0.5�1 mL depending on pellet size]. Incubate in a 65°C water bath until dissolved with gentle inversion every 30 to 60 minutes or until DNA is dissolved [may take several hours].
- Centrifuge 10 minutes in microcentrifuge at 13,000 rpm to remove any material that does not go into solution.
- Determine the DNA concentration by separating a 1:20 dilution [1 DNA:20 TE:1 loading buffer solution] on an agarose gel along with markers (uncut lambda DNA at concentrations of 10, 20, 50, and 80 ng). Stain gel with 0.5 μg/ml of ethidium bromide for 15 minutes and photograph. Visually compare the intensity of the DNA bands with the markers to determine the concentration.
