35S-RIBOPROBE SYNTHESIS FOR ISOTOPIC In Situ HYBRIDIZATION

Pipet 12.5µl 35S-UTP (1200 Ci/mmol) into 1.5ml microfuge tube. Final concentration should be 12µM. Lyophilize in speed vac. (Do not use 35S-UTP that has been previously thawed. Order 250µCi vials and use up at first thawing or discard excess via radioactive disposal.) To tube with the dried probe add:

2.0µl 5x Transcription buffer 1.0µl DTT, 100mM 1.0µl RNasin 1.0µl DNA (linearized plasmid 1µg/µl) 2.0µl GTP+CTP+ATP Mix (stock solution containing 2.5mM @) 2.0µl Sterile dH2O

Mix thoroughly and centrifuge. Add 1.0µl 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:

Vortex Add 1.0µl RQ1 DNase to the transcription reaction above Incubate 15 min., 37°C.

To extract RNA after DNase step, add to the reaction: 20µl 1x TE 1.0µl tRNA (50mg/ml) Vortex

Equilibrate 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 1µl from reaction and place in microfuge tube. Add 99µl 1xTE. Pipet 1.0µl of 1/100 dilution onto a small piece of DE81 ion exchange paper Wash 3 times 5 min. in 0.5M NaPO4, pH7.4 Wash 10 seconds in dH2O Rinse briefly (<10sec) in 100% etoh Dry throughly and count in 10ml scintillation fluid

To the riboprobe add 1xTE to a final concentration of 300,000 CPM/µl. Use immediately for in situ hybridization or store 100µl 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