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.
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