Genomic DNA Labeling Protocol

Wetypicallyuse0.5ugofE.coligenomicDNAinalabelingreactionforeachhybridization.ThegenomicDNAwasfragmentedto500to1000bpsbeforelabeling.Thefollowingprotocolshouldproduceenoughlabeledprobefor8hybridizations.

Heatto95Cfor5min,placeonicefor5min

Labeling

GenomicDNA	1.9ug/ul	2.1ul
RandomHexamer	5mg/ml	1ul
H2O		14.9
Total		20ul

Incubateat37°Cfor3.5hours

Add2.5ul0.5MEDTAtostopreaction

CleanupLabeledProbes

• PrewashMicrocon-30microfilterbyadding450mlmiliQH₂Oandspinningfor10min.@12,000RPM.
• Add450mlmiliQH₂Otoeachoftheprobesamples(ortotal500ul).�Mixthoroughlybypipettingupanddown.�TransfersamplestoseparateMicrocon-30microfilters.(Amicon)
• Spinat12,000RPMinmicrofugefor10minutesoruntil20-40mlremainsinthefilter.
• Add450mlmiliQH₂Ototheprobeandgentlymixbypipettingupanddown.�Becarefulnottotouchthefilteratthebottomofthefiltrationunit.
• Spin10minutesat12,000RPM.
• Repeatstep4,spin12mintogetsmallervolume.
• Invertcolumnintoafreshtubeandspin1minuteatmaximumspeedtorecoverprobe.�Carefullymeasurerecoveredprobevolumeifnecessary.

Probecanbestoredat4°Cor-20°Cindarkforfurtheruse.

ReagentsandSuppliers

DNAMix		20ul
dAGC	5mMeach	5ul
EcoPolBuffer	10x	5ul
CyDye-dUTP	1mM	2ul
H2O		17ul
KlenowFragment	50u/ul	1ul
Total		20ul

Cy3-dUTP	1mM	Perkinelmer	NEL578
Cy5-dUTP	1mM	Perkinelmer	NEL579
KlenowFragment	50U/ul	NEB	M0210M
100mMdNTPset*	10X	Amersham	27-2035-01
pd(N)6SodiumSalt(Hexamer)	50U	Amersham	27-2166-01
MicroconYM-30column		Amicon	42410