DNA Extraction from Rice Leaves

DNA extraction from rice leaves

Procedure:

1. Collect 1 to 3 grams of rice leaf tissue, grind in liquid N2 and transfer fine powder to a 50 ml centrifuge tube

2. Immediately add 20 ml CTAB buffer and mix thoroughly

3. Incubate at 65°C for 2 hours

4. Add equal volume of chloroform and invert tube several times to mix thoroughly

5. Centrifuge at room temperature at 10,000rpm for 10 min

6. Transfer supernatant to new centrifuge tube

7. Add equal volume of isopropanol, invert tube gently several times

8. Incubate at –20°C for 30 min or overnight

9. Spool out DNA fiber with pasture pipette hook or centrifuge at 10,000 rpm for 15 min to collect pellet

10. Discard supernatant and wash DNA pellet with 70% ethanol

11. Centrifuge at 7,500 rpm for 5 min

12. Discard supernatant and let pellet dry for 10-15 min

13. Resuspend DNA with Water or TE

CTAB buffer

25 g Sorbitol

10 g Sarkosyl (N-lauryl sarcosine)

8 g CTAB

47 g NaCl

8 g EDTA.Na2

10 g PVPP (Sigma P6755) (Polyvinylpyrrolidone)

Adjust volume to 1 liter with ddH2O

Miniprep for rice DNA extraction

Procedure:

1. Cut 1-2 young leaves and grind in liquid N2

2. Transfer powder to 1.5 ml centrifuge tube

3. Add 0.6 ml CTAB buffer and incubate at 65°C for 15 min

4. Spin for 5 min at 13,000 rpm

5. Transfer supernatant to new centrifuge tube

6. Add 0.6 ml chloroform and shake vigorously

7. Spin for 5 min at 13,000 rpm

8. Transfer supernatant to new centrifuge tube

9. Add 1.0 ml of ice cold 100% ethanol

10. Keep in –20°C for 20-30 min

11. Spin for 5 min at 13,000 rpm

12. Discard supernatant

13. Wash with 75% ice cold ethanol

14. Spin for 1 min at 13,000 rpm

15. Discard supernatant and let pellet dry

16. Resuspend DNA in 30 ul TE or water