Ribonuclease Protection Assay

1. Add appropriate volumes of RNA (<12 ul) plus probe (< 3 ul).

Try 5 and 10 ug RNA plus 600 or 800 pg dig labelled probe.

2. Include 2-3 yeast RNA samples/probe used. 2ul of 5mg/ml stock.

3. Add 20 ul of Soln. A to all of the tubes (F.V.= 30 ul) vortex and spin down.

4. Incubate at 90-95'C for 5 minutes to denature. Ice then spin down.

5. Incubate O/N at 42-45'C in a dry incubator or a water bath.

6. RNase digestion: Dilute Soln. R in Soln. Bx 1:100. Add 200 ul to each sample.

Make a 1:300 dilution by adding 66.7 ul of above to 133.4 ul of Soln. Bx.

Add Bx alone to one yeast RNA sample.

7. Vortex all samples then spin down.

8. Incubate at 37'C up to 30 minutes. Vortex then spin down.

9. Add 300 ul of Soln. Dx. Vortex then spin down.

10. Store at -20'C for  15 minutes.