Quick DNA Prep - Used for TAIL PCR
- 1. Excise 4-10 green leaves from mature plant and collect in a 1.5 ml microfuge tube.
- 2. Macerate tissue with plastic mini pestle.
- 3. Add 200 µl extraction buffer (200 mM Tris pH 7.5, 250 mM NaCl, 25 mM EDTA pH 8.0, 0.5% SDS).
- 4. Macerate again until all leaf tissue is ground up.
- 5. Vortex 5 sec. At this point, samples can sit at room temperature while remaining samples are prepared.
- 6. Spin samples 3 min at 14K in microfuge.
- 7. Transfer 150 µl of supernatant to a new tube containing 150 µl isopropanol.
- 8. Mix samples briefly.
- 9. Leave at room temperature 2 min.
- 10. Spin 5 min at 14K in microfuge.
- 11. Pour off supernatant.
- 12. Speedvac pellet for 5 min (or until dry).
- 13. Resuspend pellet vigorously in 200 µl sterile water. Use pipet tip to break up the pellet. Not all of this pellet goes into solution.
- 14. Spin 1 min at 14K to pellet junk. The pellet material will inhibit the PCR amplification so transfer the supernatant to a new tube after this spin.
- 15. Use 1µl of this DNA in TAIL PCR reactions.
- 16. Store DNA at -20°C.
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