bisufite treated products - (Aug/03/2005 )

I have done bisulfite treament several times and it works fine. But recently i have done three times, after i resuspend the pellet in water, i still can see big chuncky stuff on the bottom even after i pipet several times. And MSP showed the bisulfite reatment was bad since very low dna concentration left. Who can tell me what are those stuff remained at the bottom of the water?

Which kind of method are you using? If it is Chemicon kit, then...you might have to think about the kit's problem. If it is not, I will shut up

it could be a combination of things, your Msp primer set may not be optimal,

also i would be interested to find out what bisulfite treatment method you are using. The white stuff could be excess salts, that should go away after a 70% ethanol wash of the pellet.

Nick

This is the method i used, and i use the same primer and condition for previous bisulfite treated sample, and get really good results on real time pcr. SO I think those newly treated sample with undisolved white stuff have very low DNA concentration.

Bisulfite Treatment of DNA

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°C 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 H2O, 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°C 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°C).
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 NH4Ac, and 3 volumes of ethanol.
19. Precipitate DNA as normal (overnight at -20°C, 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°C)


*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. 89:1827-183

And if those are excess salt, how can't it disolve in the water?

Could well be excess glycogen, though I have to say I am not sure as I have never seen this before.

After precipitation did you get quite a large pellet? If so that is an indicator that it's excess glycogen. You can try and heat your sample to 50C for 15 minutes to see if it goes away. I don't think the white stuff would affect your PCR though.

The only thing I would suggest other than this, is to perform the bisulfite treatment again.

Nick