KC Lab Protocol for the preparation of riboprobe

1. Digest plasmid DNA (10 ug) with the appropriate restriction enzyme at one end of the insert

2. Check DNA digest on agarose gel for complete digestion of the plasmid

3. If digestion is completed, phenol/chloroform extraction once, ethanol precipitation and resuspend the template in 10 ul of DEPC water

4. Mix the following components for in vitro transcription at room temperature

5X Transcription buffer	4 ul
0.2 M DTT	1 ul
NTP	2 ul
DNA template (1 ug)	1 ul
35S-UTP	10 ul
RNasin	1 ul
RNA polymerase	1 ul
Total	20 ul

5. Add 2 ul of DNase I (BM, RNase-free), incubate 37oC, 45 minutes.

6. Stop reaction by adding 20 ul of column buffer and 1 ul of yeast RNA

7. Prepare gel filtration column by adding G-50 bead in short Pasteur pipette

8. Wash column twice by column buffer

9. Load 50 ul of yeast RNA into the column

10. Add probe

11. Elute with 200 ul of column buffer for 7 times at room temperature ** collect each fraction (except fraction 1 and 2) in separate Eppendorf tubes

12. Take out 1 ul from fraction 3 to 6 for scintillation counting (into 5 ml of Bray’s solution)

13. Scintillation counting (with Beckman LS 6500)

• Put the scintillation vials from left to right into the sample racks (blue or white in colour)
• Insert the program number card (no. 11) at the back of the first rack
• Place the rack on the right side of the sample changer, inserting it at the angle so that the lip on the left end slides under the groove along the middle of the sample changer
• Place additional racks if necessary and arrange as follows
• Place the red rack with halt command card in the last position
• Select Main menu / Automatic counting / Program 11 (1 minute counting for 35S)

14. Precipitate the fraction with the highest activity by ½ volume of 6 M NH4Ac and 2.5 volume of ethanol, overnight at -20oC

15. Probe spun down in microfuge at 13,000 rpm, 20 minutes, 4oC

16. Wash pellet once by 80% RNA ethanol

17. Dry pellet by inverting tube on bench for 10 minutes or using Speedvac

18. Resuspend probe in 0.1 M DTT at concentration of 2,000,000 cpm/ul

19. Dilute appropriate amount of probe with 9 volumes of hybridization mix

20. Probe (include undiluted and diluted) should be kept in -70oC freezer