Hello Yuki Yang

Have you tried using TRIzol cell lysis solution? It is composed of phenol and guanidine thiocyanate in a mono-phase solution, used for the simultaneous isolation of RNA, DNA, and proteins from sample. The chemical make-up of TRIzol facilitates the release of RNA and DNA from hard-to-lyse cells and tissue samples.

The protocol in brief:

1. For 10ml of culture, you may use 1ml of TRIzol for lysis. Incubate in TRIzol for 5 mins at room temperature to permit complete dissociation of the nucleoprotein complex.

2. Add 200ul of chloroform per 1mL of TRIzol, cap sample tube securely and shake vigorously by hand for 15 seconds. Incubate the sample at room temperature for 3 minutes.

3. Following incubation, centrifuge the sample at 12000 x g for 15 minutes at 4°C.

4. Following centrifugation, the mixture separates into a lower red phenol chloroform phase, an interphase, and a colorless, upper aqueous phase. RNA remains exclusively in the aqueous phase. However, the organic phase may be saved for subsequent DNA and protein extraction.

5. You may remove the top aqueous phase for RNA extraction (and follow the protocol for RNA extraction).

6. Take the bottom organic phase for DNA Extraction (be careful, no aqueous phase should be removed).

7. Add 400ul of 100% ethanol per 1ml of TRIzol. Cap the tube, mix by inverting the tube several times.

8. Incubate for 2–3 minutes.

9. Centrifuge for 5 minutes at 2000 × g at 4°C to pellet the DNA.

10. Transfer the phenol-ethanol supernatant to a new tube. The supernatant may be used for protein isolation, if you are interested.

11. Resuspend the DNA pellet in 1 mL of 0.1 M sodium citrate in 10% ethanol, pH 8.5.

12. Incubate for 30 minutes. Mix occasionally by gentle inversion.

13. Centrifuge for 5 minutes at 2000 × g at 4°C.

14. Discard the supernatant.

15. Repeat steps 11- 14 once.

16. Resuspend in 1.5 - 2.0ml of 75% Ethanol and incubate for 10-20 mins at room temperature. Mix periodically by gentle inversion.

17. Centrifuge for 5 minutes at 2000 × g at 4°C.

18. Discard the supernatant and air-dry the DNA pellet for 5–10 minutes.

19. Resuspend the pellet in 8mM NaOH by pipetting up and down. Resuspending the DNA in a mild base is recommended because isolated DNA using the TRIzol method does not resuspend well in water or Tris buffer.

20. Centrifuge for 10 minutes at 12,000 × g at 4°C to remove insoluble materials.

21. Transfer the supernatant to a new tube, then adjust the pH as needed with HEPES.

Hope this helps!

Good Luck!