Pipet 12.5 l 35S-UTP (1200 Ci/mmol) into 1.5ml microfuge tube. Final concentration should be 12 M. Lyophilize in speed vac. (Do not use 35S-UTP that has been previously thawed. Order 250 Ci vials and use up at first thawing or discard excess via radioactive disposal.) To tube with the dried probe add: 2.0 l 5x Transcription buffer1.0 l DTT, 100mM1.0 l RNasin1.0 l DNA (linearized plasmid 1 g/ l)2.0 l GTP+CTP+ATP Mix (stock solution containing 2.5mM @)2.0 l Sterile dH2OMix thoroughly and centrifuge.Add 1.0 l RNA Polymerase (SP6, T7 or T3 as required)Mix gently by pipeting, do not vortex, and incubate 1-2 hours, 37°C.(Order all of the above as ready-made stocks from Promega Biotech).To stop the reaction: VortexAdd 1.0 l RQ1 DNase to the transcription reaction aboveIncubate 15 min., 37°C.To extract RNA after DNase step, add to the reaction:20 l 1x TE1.0 l tRNA (50mg/ml)VortexEquilibrate one QuickSpin G-50 Sephadex column (Boehringer Mannheim Catalog No. 100-616) to room temperature for each riboprobe (15-30 min. at room temperature). Invert column gently for 20-25 times to suspend the gel.Remove TOP cap first, followed by the bottom cap.Allow the fluid within the column to drip through by gravity.Cut a collection tube (comes with column) approx. 5mm from bottom, place column in the collection tube and place the assembly in a 15ml tube.Spin for 2 min at 1100g or 2500rpm (setting #6 on IEC centrifuge).Discard fluid and collection tube.Place the column in a new collection tube, add riboprobe to the CENTER of the column. Place the assembly gently into the 15ml tube and spin for 4 min at 1100g (#6 on the IEC). Remove the assembly gently with forceps, and measure the volume of riboprobe. To monitor incorporation: Take 1 l from reaction and place in microfuge tube.Add 99 l 1xTE.Pipet 1.0 l of 1/100 dilution onto a small piece of DE81 ion exchange paperWash 3 times 5 min. in 0.5M NaPO4, pH7.4Wash 10 seconds in dH2ORinse briefly ( 10sec) in 100% etohDry throughly and count in 10ml scintillation fluidTo the riboprobe add 1xTE to a final concentration of 300,000 CPM/ l. Use immediately for in situ Hybridization or store 100 l aliquots at -70°;C up to one week in TE. Just prior to starting the hybridization step: Thaw the riboprobe and keep on ice.Make up Hybridization Mix as described in the in situ hybridization protocol