1. Cut 10ug of plasmid DNA where you want the transcript to end.

2. Add an equal volume of 2X PK buffer:

200mM TrisHCl pH 7.5

25mM EDTA

300mM NaCl

2% SDS

200ug/ml proteinase K

3. 37� for 30min and then extract with phenol/CHCl3 and ethanol precipitate. Resuspend at 1mg/ml in DDW.

4. Labeling reaction:

2ul 5X SP6 buffer: 200mM TrisHCl, 30mM MgCl2, 10mM spermadine

0.5ul RNAsin

0.5ul 10mM rATP

0.5ul 10mM rCTP

0.5ul 10mM rGTP

5ul a-P32-UTP (400Ci/mMol)

0.5ul template

0.5ul 200mM DTT

0.5ul appropriate RNA polymerase

Incubate for 90-120min at 41�.

5. Add 0.5ul RNAsin and 0.5ul RNAse free DNAse (5mg/ml). Incubate for 15min at 37�.

6. Add 10ug tRNA and 100ul DDW. Phenol/CHCl3 extract and precipitate with 50ul 5M NH4 acetate, 400ul ethanol.

7. Resuspend in 100ul 1X hybridization buffer:

0.1 volume 10X hyb. (400mM pipes, pH 6.4, 4M NaCl, 10mM EDTA)

0.1 vloume DDW

0.8 volume deionized formamide.

1ul should be 1000-2000 cps on the mini monitor.

8. Spin down RNA as an ethanol ppte and resuspend in 24ul 1X hyb. buffer and add 0.5-1.0ul of probe (from step 7). Include a control RNA eg. tRNA or negative tissue.

9. Incubate overnight at 37?50�.

10. Add 350ul RNAse buffer to hybridization and vortex immediately.

1X RNAse buffer:

10mM Tris pH 7.5

5mM EDTA

300mM NaCl

40ug/ml RNAse A (Sigma)

2ug/ml RNAse T1 (Sigma)

11. Incubate at ___ � for ____min.

Optimal times and temperatures vary for different probes. Overdigestion gives lots of smlall bands and underdigestion gives large artifactual bands. 37� for 10-15min works well in many cases.

12. To stop the reaction add 20ul 10% SDS and 5ul 20mg/ml proteinase K. Incubate for 15min 37�.

13. Add 2-10ug tRNA carrier. Phenol chloroform extract and ethanol preciptate without additional salt. Air dry pellet and resuspend in 3-4ul sequencing loading buffer and run on a sequencing gel after boiling.