FLORACLEAN
Plant Genomic DNA Isolation Protocol
Revision No. 2012-999-5L01W

The kit is shipped at ambient temperature. However, upon arrival, store Protease Mixx at 4°C. A few days at ambient temperature does not affect activity of this reagent. A precipitate (often appears fungus-like) is sometimes evident after storage. Remove through centrifugation. Protease activity is not affected.

The FLORACLEAN Plant DNA Isolation Kit is used to efficiently isolate high molecular weight genomic DNA from a wide range of plant tissues and species. Each preparation yields up to 400 g of genomic DNA from 1.5 g of starting tissue. Preparation sizes can be scaled up or down. The DNA is isolated from purified nuclei, thus eliminating many cytoplasmic compounds that copurify with DNA when using other methods of DNA isolation and which can inhibit enzymes. The procedure requires about 2 hours of manipulation and 2-4 hours of incubation time. Resulting DNA is suitable for restriction enzyme digestion or PCR amplification. The procedure does not use organic extractions.

The FLORACLEAN Plant DNA Isolation Kit is available in 3 sizes to suit your needs: 10, 25, and 100 preparations/kit. Each kit contains Nuclear Buffer for lysing cellular membranes, chloroplasts, and mitochondria while keeping nuclei intact. Nuclei are lysed with Nuclear Lysis Solution. The kit utilizes Protease Mixx to degrade cellular proteins. This is followed by a proprietary "salting-out" technique which precludes the need for phenol, chloroform, or other organic extractions. The DNA is precipitated with ethanol, pelleted by centrifugation, and dissolved in TE.

Prepare Tissue

1. Grind tissue in liquid nitrogen (optional).

Lyse Cellular Membranes

2. Homogenize 1.5 g tissue in 10 ml of Nuclear Buffer.
3. Filter homogenate through Sieve Material.

Isolate and Wash Nuclei

4. Pellet nuclei at 2000 x g for 20 minutes at 4°
5. Wash nuclei 3 times with 5 ml of Nuclear Buffer.

Lyse Nuclei

6. Resuspend washed nuclear pellet in 1.5 ml of Nuclear Suspension Solution.
7. Lyse nuclei by addition of 100 µl of Nuclear Lysis Solution.
8. Add 25 µl of Protease Mixx and incubate at 55°C for 1-4 hours.
9. Add 500 µl of "Salt-Out" Mixture; ice 15 minutes; pellet and discard precipitate.

Precipitate DNA

10. Add 2 ml water and 8 ml ethanol to precipitate DNA and centrifuge.
11. Dissolve DNA pellets in 100-200 µl of water or TE.

Prepare Tissue


1. Grind tissue to a fine powder with a mortar and pestle or Waring blender in liquid nitrogen. Allow the liquid nitrogen to evaporate (tissue becomes lighter in color and does not form clumps); however, do not allow the tissue to thaw. Tissue powder can be stored at -70°C indefinitely.




Fresh tissue can also be homogenized without the need to grind if the tissue is relatively soft [young leaves or flowers, e.g.]. The results are equivalent to those where prior grinding is included.



Lyse Cellular Membranes


2. Add 1.5 g ground, frozen plant tissue (see Note below) to a 40 ml Dounce (or equivalent) homogenizer on ice; add 10 ml of cold Nuclear Buffer and homogenize until the "tight" plunger smoothly passes by the tissue (usually 4-10 passes of plunger past tissue; clearance between "tight" plunger and wall of homogenizer is enough to allow nuclei to remain intact). The homogenizer can be removed from the ice for a minute or two while this is being done.
3. Fold a 4x4 inch square of Sieve Material (cut from the material supplied) twice to form a 2-inch square, four layers thick. Separate one of the layers with gloved fingers; place in a small funnel and pour homogenate through the conical sieve allowing the liquid to pass into a beaker on ice. Do not squeeze the last bit of liquid out of the retained debris. Discard the tissue pieces retained by the sieve. Rinse and blot the sieve fabric for reuse.


Isolate and Wash Nuclei


4. Pour the strained liquid into centrifuge tubes or bottles and centrifuge at 2,000 x g for 20 min at 4°C to pellet nuclei. Discard the supernatant fractions which should be green due to lysis of chloroplasts.
5. Resuspend the nuclei in 5 ml of cold Nuclear Buffer either by hand or gentle vortexing. Pellet nuclei as in step 4. Repeat this wash twice. After that, the nuclear pellet should be grayish-white and not very viscous. Chloroplast or starch contamination causes a green or viscous nuclear pellet. Cells from some tissues will not lyse as easily as others and the pellet will have a greenish tint but low viscosity. DNA isolated from these pellets appears to be of good quality and will readily cut with restriction enzymes.


Lyse Nuclei


6. Resuspend the washed nuclear pellet in 1.50 ml of Nuclear Suspension Solution and transfer to a 2 ml microcentrifuge tube.
7. Add 100 µl Nuclear Lysis Solution and incubate at 55°C for 30 minutes.
8. Add 25 µl of Protease Mixx and continue incubation at 55°C for 1-4 hours. (The longer times increase the probability of complete digestion of protein, including self-digestion of protease).
9. Add 500 µl "Salt-Out" Mixture (split into two microcentrifuge tubes if necessary) and incubate on ice for 15 minutes (or overnight in refrigerator). Centrifuge at high speed in microcentrifuge for 5 minutes to pellet precipitated debris.


Precipitate DNA


10. Transfer supernatant (pool if in two tubes) to a 15 ml polypropylene or glass centrifuge tube with screwcap lid. Add 2 ml water; mix, and add 8 ml 100% ethanol. Mix by inverting.
    1. If stringy white DNA precipitate becomes visible, centrifuge for 20 minutes at 2000 x g in a benchtop clinical centrifuge equiped with a swinging bucket rotor to pellet precipitated DNA. Discard supernatant; blot walls of tube,; and air dry pellet for 15 minutes. (To assist in dissolving DNA, do not dry pellet under vacuum).
    2. If stringy white precipitate is not evident place tube at -70°C for 20 minutes (or -20°C overnight) and centrifuge at 10,000 x g (preferably in a swinging bucket rotor) for 20 minutes. Discard supernatant and air dry pellet as in step a, above.
11. Dissolve DNA pellets in 100-200 µl of autoclaved water or TE at room temperature, or overnight at 4°C.

DNA isolated from tissues of most species, by the above procedure, will digest easily with restriction enzymes and/or amplify. If, however, a DNA Genomic DNA Isolation from Whole Blood sample is resistant to enzymes, an additional cleaning step with GENECLEAN SPIN (see pg 18) will invariably reverse the inhibition.