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°C for 30min and then extract with phenol/CHCl3 and ethanol precipitate. Resuspend at 1mg/ml in DDW.
4. Labelling 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°C.

5. Add 0.5ul RNAsin and 0.5ul RNAse free DNAse (5mg/ml). Incubate for 15min at 37°C.
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 volume 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°C.
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 ___ °C for ____min.

Optimal times and temperatures vary for different probes. Overdigestion gives lots of small bands and underdigestion gives large artifactual bands. 37°C 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°C.
13. Add 2-10ug tRNA carrier. Phenol chloroform extract and ethanol precipitate without additional salt. Air dry pellet and resuspend in 3-4ul sequencing loading buffer and run on a sequencing gel after boiling.