We also use other extraction methods: Phenol/Chloroform
2. Rapidly wash tissue with 1ml of cold STE buffer two times. Remove the wash liquid with a P1000 pipetman.
3. Add 550ul tissue lysis buffer and 11ul proteinase K. Incubate at 60°C for 5 hours to overnight. (Additional proteinase K can be added if the tissue is still solid following incubation, or a gentle vortex may break up remaining tissue).
4. Remove from incubator and add 5.5ul RNase A (10mg/ml).
5. To precipitate proteins from tissue solution, add 350ul 5M sodium chloride. Mix well by inverting tube. Centrifuge in the microcentrifuge for 30 minutes @ 14000 RPM (total volume is about 900ul)
7. Transfer approximately 1/2 the supernatant (410-420ul) to each of two (2), 1.5ml tubes. The pellet contains proteins and other cellular debris which can be disposed of.
8. Add 900ul of 100% cold, absolute ethanol to each tube. Invert tube gently several times. At this point you may be able to see the DNA as a clearish, white precipitate.
9. Incubate samples at -20°C for 2 hours to overnight.
10. To pellet the DNA, centrifuge samples for 30 minutes @ 14000 RPM in the microfuge.
11. Pour off the supernatant carefully. Be careful not to disturb the DNA pellet. Remove excess ethanol by touching the edge of the microcentrifuge tube to a clean Kimwipe.
13. Add 1ml cold 70% ethanol remove excess salts from the DNA pellet. Once again, be careful not to disturb the DNA pellet.
14. Microcentrifuge for 5 minutes at 6000 RPM.
15. Gently pour off the 70% rinse ethanol.
16. Kimwipe the edge of the tube to remove excess ethanol.
17. Repeat steps 13 - 16 again (optional).
18. Cover the Eppi tubes with a Kimwipe and place in 37°C incubator until dry. -OR- place Eppi tubes in Speed-Vac to dry.
19. Suspend DNA pellet in 50-100 ul of T(1/10)E (or other EDTA-based buffer) and incubate at 60°C for 1 hour (People usually combine the two tubes from Step 7 at this point for a total volume of 100-200 ul)
20. Label the sample and store at -20°C.
21. Proceed to PCR
A 1/100 dilution (or 1/10, 1/50, etc.) of the above
DNA solution will sometimes result in more successful PCRs (see PCR protocols).
Suspend 5ul DNA in 495ul distilled water and label appropriately.