57. Preparation of DNA from single rice seedlings

C. Have a supply of broken tip pasteur pipets (use a file to score each pipet and break off the tip to leave an opening of about 3 mm), pipet bulbs, microfuge tubes, 20 u1 and 200 u1 pipetting devices.

D. Aliquot all solutions into tubes and place on ice. Never pipet directy from the stock bottles.

G. Make a sheet listing the basic steps of the protocol and all of the sample numbers: use this list to check off that you have completed each step for each sample.

Required solutions: Make one day in advance. All store well for many months. 20% SDS should be stored at room temperature; all of the other solutions should be stored in a refrigerator.

Solution 1: 15% sucrose, 50 mM Tris-HC1, pH8, 50 mM Na\2\EDTA, 250 mM NaC1 -autoclaved.

We keep seeds in labeled envelopes. Prior to planting, each type of seed is soaked separately in a small beaker with distilled water for 30minutes. Then soak the seeds for 20 minutes in 0.5% hypochlorite bleach. Rinse seeds thoroughly in sterile water.

1. This method is appropriate for small samples of seedlings; see the rice mitochondria protocol for details on large scale germination. Label edge of plastic tray with seedling numbers. Fill tray with 2.5 cm of vermiculite and put seed on top. Soak vermiculite with distilled water and a pinch of fungicide. Germinate seedlings for about 4-6 days in 25 degrees C room in dark, and then harvest when about 1-3 cm long. Cut the seedlig top at the joint with the seed coat. Place clipped seedlings into the seed envelope they came from. Put the envelopes in -80 degrees C freezer for storage.

2. Move envelopes to -20 degrees C freezer near your work area. Remove one seedling and homogenize it in cold mortar by rapid grinding, then add 0.5 ml Solution 1 and grind again. Using a broken tip pasteur pipet, transfer the fluid to a microfuge tube sitting on ice. Rinse mortar twice with 0.5 ml Solution 1 to pick up resideu; regrind if you see solid material. Close the lid of the microfuge tube and check to make sure that it is properly labeled. If the same genotype sample is used next, wipe mortar and pestle dry before next seedling; if changin genotype, wash and dry thoroughly. Keep sample on ice.

3. When 12-18 samples are ready, spin the microfuge tubes at low speed in a clinical centrifuge for 5 minutes in the cold room. For the Clay-Adams clinical centrifuge, use a setting of 60 for 5 minutes.

4. Remove and discard the supernatant with a pasteur pipet-do this carefully because the pellet is very soft. The pellet contains "crude nuclei". Resuspend the crude nuclear preparation in 0.3 ml Solution 2. Tap the bottom of tube gently to get the pellet in solution. Then add 20 ul of 20% SDS and mix again by inverting the tube rapidly several times.

6. Remove tubes from heat and add 150 ul of 7.5 M ammonium acetate and mix thoroughly by inverting the tube several times.

8. Sediment the ammonium acetate-SDS precipitate at high speed in a microfuge in the cold room for 5 minutes.

9. Using a cut plastic pipet tip or broken pasteur pipet, transfer the supernatant to a new microfuge tube, add 0.7 ml isopropanol and let sit at 15 minutes. High molecular weight DNA will form "strings" when mixed with isopropanol; the higher the molecular weight of the DNA, the more air bubbles will be trapped in the strings. Sediment the DNA in the cold room in a microfuge for 5 minutes. High quality DNA will form a translucent, gray-white pellet. Bright white pellets contain carbohydrate; green pellets contain chlorophyll.

10. Decant the supernatant and invert tubes on paper towel for a few minutes. Wash the DNA pellet in 1 ml cold 75% ethanol. Microfuge again in cold for five minutes and pour off the ethanol. Dry in vaccum for about 15 minutes or in air for about 20 minutes. Resuspend in 100 ul TE overnight at 4 degrees C.

11. DNA yield can be checked by comparison to known standards: (1) by gel electrophoresis, (2) by mixing 1 u1 of your sample with a small amount of ethidium dye on a piece of parafilm and comparing to the fluorescence from 1 ng, 3 ng, 10 ng, and 100 ng of DNA, or (3) by spectrofulorimetry. Usual yield ranges from 3 to 10 ug per seedling, or about 0.03-0.1 ug per u1 if the final preparation is resuspended in 100 u1. DNA should be larger than 20 kb.

It is convenient for one person to do 12 samples. These will take about 30-40 min to grind and the rest of the procedure requires less than 2 hr. Scale up to 18-48 samples if you can get some assistance with the initial grinding step.

If RNA is a problem for subsequent use of samples, RNase treat after the first resuspension for 1 hour at 37 degrees C. Extract with buffered phenol, and then re-extract the aqueous phase with chloroform twice. Then add 1 volume of ethanol, freeze the sample to preciptiate the DNA in the cold for several hours. Spin in microfuge, and resuspend pellet in 100 u1 of TE.