--Karen Cone

Last year, during a mutant hunt I was faced with the prospect of doing hundreds of DNA preps for the purpose of isolating enough DNA for one or two Southerns. I modified a large-scale method we had been using for leaf DNA preps. There are two main advantages to this method. All centrifugations are performed in microfuge tubes, thus facilitating the handling of large numbers of samples in a short period of time. The inclusion of a phenol/chloroform extraction eliminates most nuclease activity, and therefore the DNA is stable for fairly long periods at -20 C.

10 X lysis buffer: 3.5M NaCl; 0.01M Tris-HCl, pH 7.6; 0.01M EDTA.

1X final lysis buffer is 1X lysis buffer, 7M urea, 2% sarkosyl, 50mM EDTA.

For 100ml, 10ml 10 X lysis buffer, 42g urea (ultrapure), 10ml 20% sarkosyl, 10ml 0.5M EDTA, pH 8.0.

1. Harvest piece of leaf from seedling (usually at the 3-4 leaf stage). Leaf piece should weigh not more than 0.3g (about 3" x 3/4 " is right size). Roll up and put into 15ml polypropylene tube.

2. Pour liquid nitrogen into tube and push leaf to bottom to freeze. Grind to fine powder with glass rod. Keep on dry ice till all samples are processed.

3. Add 0.6ml lysis buffer. Warm in 42 C bath till thawed.

4. Shake in 37 C bath for 10 min.

5. Add 0.5ml phenol:chloroform:isoamyl alcohol (100:100:1). Vortex gently 30 sec. Shake at 37 C for 10 min.

6. Transfer tube contents to 1.5ml microfuge tube. Spin 5 min in microfuge.

7. Remove 500ul supernatant to new microfuge tube. Add 50ul 3M NaAc, pH 5 and 600ul isopropanol. Invert several times to mix.

8. Spin in microfuge for 1 min. (no longer or pellet will be hard to dissolve). Remove supernatant with drawn-out pasteur pipette.

9. Add 500ul 70% ethanol. Spin 30 sec. Remove supernatant with drawn-out pipette.

10. Resuspend pellet in 100ul TE. Vortex gently till dissolved--should take about 30 min with intermittent vortexing.

11. Store at -20 C.

Use 5-10ul per restriction digest. Addition of spermidine (to a final concentration of 4mM) to restriction digests facilitates complete digestion. When loaded in 2.5mm wells, blotted to nitrocellulose, and probed with single-copy hybridization probes, the time required for proper exposure is 1-2 days with Lightning-Plus intensifying screen and XAR film at -80 C.

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