Adapted from Frommer et.al.*

1.Dilute DNA (up to 2 mg) into 50 ml with distilled H₂O.

2.Add 5.5 ml of 2M NaOH.

3.Incubate at 37℃ for 10 minutes (to create single stranded DNA).

4.Add 30 ml of 10 mM hydroquinone (Sigma) to each tube, freshly prepared by adding 55 mg of hydroquinone to 50 ml of water.

5.Add 520 ml freshly prepared 3M Sodium bisulfite (Sigma S-8890), prepared by adding 1.88 gm of sodium bisulfite per 5 ml of H2 O, and adjusting pH to 5.0 with NaOH.

6.Assure that reagents are mixed with DNA.

7.Layer with mineral oil.

8.Incubate at 50℃ for 16 hours (avoid incubations of much longer duration as methylated C will start converting to T).

9.Remove oil.

10.Add 1 ml of DNA wizard cleanup (Promega A7280) to each tube and add mixture to miniprep column in kit.

11.Apply vacuum (manifold makes this convenient).

12.Wash with 2 ml of 80% isopropanol.

13.Place column in clean, labeled 1.5 ml tube.

14.Add 50 ml of heated water (60-70℃).

15.Spin tube/column in microfuge for 1 minute.

16.Add 5.5 ml of 3 M NaOH to each tube, and incubate at room temperature for 5 minutes.

17.Add 1 ml glycogen as carrier (we use Boehringer glycogen, undiluted).

18.Add 33 ml of 10 M NH₄ Ac, and 3 volumes of ethanol.

19.Precipitate DNA as normal (overnight at -20℃, spin 30 mins), wash with 70% ethanol, dry pellet and resuspend in 20 ml water.

20.Treat DNA like RNA (keep cold, minimize freeze/thaws, store at -20℃)

PCR Tips

1.Make mix with primers, NTPs and buffer.

2.Add 2 ml of DNA per PCR reaction (50 ml).

3.Hot start is needed for PCR to discriminate unmethylated/methylated DNA.

4.Always include water control and positive controls for the PCR.

*Frommer, M., L. E. McDonald, D. S. Millar, C. M. Collis, F. Watt, G. W. Grigg, P. L. Molloy, and C. L. Paul. 1992. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci, USA. 89:1827-1831.