Quick DNA Prep - Used for TAIL PCR

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.