PreparationofFluorescentDNAProbefrommRNA:Yeast
Lastupdated:3/29/99Materialsfor2reactions(Cy3&Cy5) Qty Orderinfo Heatblocks,42C&70C 1each OligodT(2ug/uL) 5.0uL SuperscriptIIRTkit GibcoBRL#18064-014 50XdNTPs(25mM;10mMdTTP)* 1.25uL Cy3-dUTP(1mM) 3.0uL Amersham#53022 Cy5-dUTP(1mM) 3.0uL Amersham#55022 Microcon-30filter 3 Amicon#42410 TE(pH7.4) ~5mL polyADNAorRNA(10mg/mLinTE) 1.0uL 20XSSC 3.0uL Milliporefilter,0.45um 1 Millipore#UFC3OHVNB . *50XdNTPsstock Finalconc. dATP(100mM) 25uL 25mM dCTP(100mM) 25uL 25mM dGTP(100mM) 25uL 25mM dTTP(100mM) 10uL 10mM ddH2O 15uL --------- 100uL 1. PREPAREPRIMER-ANNEALEDRNA: Cy3rxn Cy5rxn RNA(2ugmRNAor15ugtotal) 12.9(RNA#1) 12.9(RNA#2) OligodT(2ug/uL) 2.5 2.5 --------- --------- 15.4uL 15.4uL Heat10min.@70C.Quickchillonice. 2. PREPARERTCOCKTAILFORBOTHRXNS: X1 X2.5 Finalconc. 5XSuperscriptIIbuffer 6.0 15.0 1X DTT(0.1M) 3.0 7.5 10mM 50XdNTPs 0.6 1.25 500uM;200uMdTTP Cy3-orCy5dUTP 3.0 --- 100uM SuperscriptII(200U/uL) 2.0 5.0 13U/uL --------- --------- 14.6uL 11.6uLaliquots 3. Add3.0uLCy3orCy5torespectiveprimer-annealedRNAs. Aliquot11.6uLofRTcocktailtoeachrxnfortotalvolumeof30uL. Incubate2hr@42C.Placeonice. 4. Place500uLTE(pH7.4)eachintwomicrocon-30filters. AddRTrxnstoeachmicroconfilter.Centrifuge7min.attopspeed. Optional:RepeatTEwashes1-2times,oruntilallunincorporateddyeisremoved. 5. Inspectfilters.Centrifugein30sec.intervalsuntilvolumeis10-20uL. 6. Invertfiltersintofreshtubes.Centrifuge1min.toharvestlabeledCDNA. Optional:Fornextdayhybridization,storecDNA@4C. 7. MixtogetherCy3-andCy5-labeledcDNAs. Concentratesampleto~10-12uLusingmicroconfilterorvacuumpump. 8. Add1uLpolyADNAorRNAfornon-specifichybridization. Add3uL20XSSCfortotalvolumeof12-15uL. 9. Pre-wetmilliporefilterbyadding5uLddH2O.Centrifuge1min.attopspeed. Removeelutedwaterwithpipettip. 10. Addprobetofilter.(Pipetprobeontofilterwall,notdirectlyontomembrane.) Centrifuge1min.attopspeed. => PROBEISNOWREADYFORHYBRIDIZATION. REMEMBERTOADD0.3uLSDS(10%)TOPROBEasstatedinhybridizationprotocol.
