Nick Translation of DNA for CGH

1.Preparereactionmixturesper50ulAddEnzymeslast,gentlyvortexmixture,quickspinliquidtobottomoftube:

Label	Biotin-dUTP	Digox-dUTP	FITC-dUTP	TexasReddUTP
10XBiotindNTP	5ul	0ul	0ul	0ul
10XA4dNTP	0ul	5ul	5ul	5ul
dUTP	0ul	1ul	1ul	1ul
DNAPOL-1	1ul	1ul	1ul	1ul
DNAPol/DNAse(additional)	(2.5-5ul)	(2.5-5ul)	(2.5-5ul)	(2.5-5ul)
DNA(1ug)
ddH2O
TotalReactionVolume	50ul	50ul	50ul	50ul

2.Incubatereactionmixturesfor60minutes*at15C(prepareinadvanceusingicebucket,waterandice).

3.Stopreactionbyheatingat70Cfor15minutes.

4.Run3-5ul*ofprobeon1%agarosegeltochecksize.Productshouldrunasasmearrangingfrom0.3-2.3kb.

5.Storeprobesat-20C.

NOTES:

Thevolumeofenzyme,timeofincubation,andamounttorunonagarosegelwillvarydependingonsampleandenzyme:

Sample	DNA	Enzyme	EnzymeVol	Time	GelVol	Probesize
Fresh:	1ug	10X	3-5ul	60-70min	5ul(12comb)	300-2.3kb(sizeiscritical)
Paraffin	1-2ug	10X	5ul	70-90min	5ul(12comb)	300-4kb
PCRAmplifiedFreshDNA	25ul	mix	3.5ul	60min	3ul(16comb)	100-2kb
PCRAmplifiedMicrodissectedDNA	40ul	mix	3.0ul	60min	3ul(16comb)	100-600bp

The10Xenzymeisthe"fastenzyme"andweorderthesamelotofthisforseveralmonths.Theslowenzymeisaseparateenzyme,whichrequirestestingonfreshPCRamplifiedDNAwithFITCandorTexasRedtodeterminewhatmixtureoffastandslowtouseforsubsequentnicktranslations.

SOLUTIONS

10XA4:

• 5ulof10mMdATP,5ulof10mMdCTP,5ulof10mMdGTP(Gibco;finalis200uM,10X)
• 125ul1MTris,pH7.2(final500uM,10X)
• 12.5ul1MMgCl2(final200uM,10X)
• 1.7ul14.7Mmercaptoethanol(100mM,10X)
• 0.5ul50mg/mlBSA(Gibco;final100ug/ml,10X)
• 95.3uldH20tofinalvolumeof250ul.
• Storeat-20Cinscrewcaptubes.

TexasRed-dUTPandFITC-dUTP:

• 25nmfromDupont/NEN(catalog#NEL413&NEL417;Remembertospindownafterthawing,beforeopening!!!).

Dig-11-dUTP

• 25nm(BoehringerMannheim);spindownbeforeopening.

DNAPol-1:

• (Gibco/BRLcat#18010-025)

DNAPolymerase/DNaseI:

The10XEnzymemixisfromtheBIONICKkit(GIBCO).IfthisenzymeiscuttingtheDNAtoosmallthentryvaryingtheamountand/ortimeofincubation.ThestandardconcentrationforfreshDNAis3ulofthe10XEnzymemixfor60minutes.Incorporationdecreasesappreciablybelow2ulorlessthan40mins.Ifthemixisstillcuttingtoosmall,withtheseminimumamounts,thentrythe"slow"enzymemix(below),oramixtureofthetwo.Weattempttofreezedownonelotof10XEnzymewithknowncharactersticstobeusedforseveralmonths.

DNAPol-1/DNaseI:thisisthe"slowenzyme"mixintheNickTranlsationKitfromGIBCO.YoucanalsoordertheenzymeseparatelyfromGibco(catalog#18162-016).Thisenzymeislessactivethanthe10XEnzymemixfromtheBioNickkit.Ifyouneedtousethisthentry60minutesusing5ulfirst,andadjustconditionsasneeded.ThisenzymeisbetterforsmallerDNAand/ordegradedDNA.FRESHDNA:Thisshouldbeofhighmolecularweight.IftheDNAhassomedegradedlowermolecularweightDNApresentthentheenzymeusedfortheNickTranslationshouldprobablybethe"slow"enzymeasdiscussedabove.PARAFFINDNA:Formalinfixeddnaappearstoberesistanttocuttingandincorporation.Itisbesttousethefull5ulof10Xfastenzymefor90minutes,unlesstheDNAisgettingcutupeasily.IfthevolumeoftheDNAinTEisgreaterthen10ul,addmoreenzymetocompensatefortheextraEDTAfromtheTEinthereaction.