Dissolveabovesolutionsbygentlyinvertingwithaminimumamountofmixing.KeepsolutionscoldandinthedarkasmuchaspossIBLe.

Procedure

1. Digest2-5µgofhigh-molecular-weightgenomicDNAovernightwitharestrictionenzymethatdoesnotdigesttheDNAwithintheregionofinterest.
2. ExtracttheDNAwithphenol:chloroform:isoamylalcohol(PCI;25:24:1),precipitatewith1/10volume5Mammoniumacetateand2volumesethanolat-85¡ãCfor15min.Centrifuge(14,000rev/min)atroomtemperature,removethesupernatantandwashthepellettwicewith70%ethanol.DrythepelletandresUSPendin100µlTE(10mMTris-HCl,pH7.5,1mMEDTA).
3. DenaturetheDNAbyaddingfreshlypreparedNaOH(3M)toafinalconcentrationof0.3M.Incubateat42¡ãCfor30min.
4. Toasiliconizedmicrocentrifugetubeadd:
   • 1020µl40.5%sodiumbisulfite
   • 60µl10mMhydroquinone
   • 110µlDNA(+NaOH)
   • 10µlwater
5. Gentlymixandoverlaywithmineraloil.Coverthetubewithaluminumfoiltoshieldfromthelight.
6. Incubateat55¡ãCfor16-18h.
7. PurifyDNAusingtheGenecleanIIkit(IntermountainScientificCorporation)oranyothermethodsofyourchoice.
8. Afterpurification,resuspendDNAandaddTEtoafinalvolumeof100µl.
9. DenaturethesamplewithfreshlypreparedNaOH(asabove)andincubateat37¡ãCfor15min.
10. Neutralizebyaddingammoniumacetate(pH7.0)to3M.
11. PrecipitatetheDNAwiththreevolumesofethanol,centrifugefor10min(14,000rev/min)atroomtemperature,washtwicewith70%ethanolanddryunderavacuum.Resuspendin50µlTE,andstoreat-20¡ãCwrappedinfoil.TreatedDNAshouldbeusedwithinonemonthasdegradationmayoccurinthecleanedandfrozensample.

BisulfitemodificationfornanogramquantitiesofDNA

FormodificationsofsmallerquantitiesofDNAsuchasDNAfrommicrodissectedtissues.Thebasicchangesthatismadetotheaboveprotocolincludethemethodofextractionandtheadditionofmusselglycogentoprecipitations.

Thestepsarethesameasaboveandonlychangesarenoted.

1. InStep4,thebisulfitereactionisscaleddowntoaccommodatethesmallervolumeofDNA:
   • 255µlsodiumbisulfate
   • 15µlhydroquinone
   • 27.5µlDNA(+NaOH)
   • 2.5µlwater
2. InStep8,TEisaddedtoafinalvolumeof25µl.
3. InStep11,theprecipitationisperformedinthepresenceof40µgmusselglycogenat-80¡ãCfor30min,followedbycentrifugation(14,000rev/min)atroomtemperaturefor30min.TheDNAisresuspendedin25µlTE.

Note

1. ItisimportantthattheDNAiscompletelydenaturedpriortoandinthepresenceofthebisulfitesolutionorthemodificationwillnotbecomplete.
2. Toensurecompletedenaturation,nomorethan5µg(orless)ofstartingmaterial(DNA)shouldbeused
3. Theinitialalkalinedenaturationshouldbeat42¡ãCfor30min.
4. TheDNAdigestionwithrestrictionenzymescanbeomitted.