|CpG Island Methylation in Aging and Cancer|
Bisulfite Treatment of DNA
Adapted from Frommer et al.*
- Dilute DNA (up to 2 mg) into 50 ml with distilled H2O.
- Add 5.5 ml of 2M NaOH.
- Incubate at 37oC for 10 minutes (to create single stranded DNA).
- Add 30 ml of 10 mM hydroquinone (Sigma) to each tube, freshly prepared by adding 55 mg of hydroquinone to 50 ml of water.
- Add 520 ml freshly prepared 3M Sodium bisulfite (Sigma S-8890), prepared by adding 1.88 gm of sodium bisulfite per 5 ml of H2O, and adjusting pH to 5.0 with NaOH.
- Assure that reagents are mixed with DNA.
- Layer with mineral oil.
- Incubate at 50oC for 16 hours (avoid incubations of much longer duration as methylated C will start converting to T).
- Remove oil.
- Add 1 ml of DNA wizard cleanup (Promega A7280) to each tube and add mixture to miniprep column in kit.
- Apply vacuum (manifold makes this convenient).
- Wash with 2 ml of 80% isopropanol.
- Place column in clean, labeled 1.5 ml tube.
- Add 50 ml of heated water (60-70oC).
- Spin tube/column in microfuge for 1 minute.
- Add 5.5 ml of 3 M NaOH to each tube, and incubate at room temperature for 5 minutes.
- Add 1 ml glycogen as carrier (we use Boehringer glycogen, undiluted).
- Add 33 ml of 10 M NH4Ac, and 3 volumes of ethanol.
- Precipitate DNA as normal (overnight at -20oC, spin 30 mins), wash with 70% ethanol, dry pellet and resuspend in 20 ml water.
- Treat DNA like RNA (keep cold, minimize freeze/thaws, store at -20oC)
- Make mix with primers, NTPs, and buffer.
- Add 2 ml of DNA per PCR reaction (50 ml).
- Hot start is needed for PCR to discriminate unmethylated/methylated DNA.
- 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 U. S. A. 89:1827-1831.