Isolation of Chloroplast DNA

1. Grind in a blender with 7 vol LL for maize, and 10 for sorghum. Use MCE at 350 g/L, and if necessary, 5 mL 1 M DIECA/L. Cut leaves into ca. 1" strips prior to blending.
2. Squeeze through cheesecloth, and Miracloth.
3. Centrifuge in GSA/JA14 rotors, if necessary. 10 min at 500 rpm GSA;10 min at 570 rpm JA14.
4. Pellet chloroplasts in GSA/JA14; 15 min at 3000 rpm GSA;15 min at 3100 rpm JA14.
5. Resuspend with pipette, in LL without MCE, etc. (ca. 300 mL/250 g).
6. Centrifuge 10 min in SS34/SA600/JA17 rotors: SS34 600 rpm; SA600 530 rpm; JA17 560 rpm.
7. Pellet chloroplasts 15 min in SS34/SA600/JA17 rotors: SS34 3000 rpm; SA600 2700 rpm; JA17 2800 rpm.
   Use of small rotors is preferable.
   
8. Resuspend with pipette in LL, ca. 250 mL/100 g centrifuge, as in step 7.
9. Resuspend with pipette in LL, ca. 10 mL/100 g if possible.
10. Add MgCl₂ to 10 mM (100 mL 1M/10 mL); DNAse to 100 g/mL (250 mL 2 mg/mL/10 mL). Incubate 60 min at room temperature.
11. Bring to about 250 mL/100 g with MM; centrifuge as in step 7; resuspend with pipette in MM to ca. 80 mL; centrifuge as in step 7.
12. Resuspend with pipette in NN; minimum volume, ca. 5 mL/100 g.
13. Add SDS to 0.5% (250 mL of 10%/5 mL NN). Swirl.
14. Add Proteinase K to 200 g/mL (500 mL of 20 mg/mL/5 mL). Swirl.
15. Lyse 60 min at 37°C. (Go to dellaporta for sorghum).
16. Maize: 3 standard phenol:chloroform extractions, ethanol precipitation.