In Vitro Transcription and S1 nuclease protection analysis of Pol I transcripts
Adapted from McStay and Reeder (1986). Cell 47:913-920.
Procedure
Prepare 2x reaction mix: 30mM HEPES pH 7.9, 80 mM KCl, 12 mM MgCl2, 1mM DTT, 200ug/ml alpha amanitin (caution: very toxic!)
To sterile 1.5 ml microfuge tubes add:
20ul cell extract or column fraction (in 100mM KCl buffer) and 1ul of template DNA (10-200 ng depending on degree of polymerase purification)
-Mix by tapping, or vortex at moderate speed (about 5-6 on scale of 10)
-incubate 5 min at room temp
-add 20ul 2X reaction mix and mix by tapping or vortexing
-incubate at 25 degrees C for 1-3 hours
Stop reactions by adding 360ul of stop buffer: 0.15M NaCl, 50mM Tris-HCl pH 8.0, 250 mM sodium acetate, 0.25% SDS, 6mM EDTA pH 8.0
- extract the stopped reactions once with 400ul of 1:1 phenol:chloroform (where chloroform is actually 24:1 chloroform:isoamyl alcohol)
-vortex 5 seconds, then spin at room temp. in microfuge for 3 min.
-remove aqeous to fresh tube containing 10-20 ul of 5’ end-labeled DNA probe
-Add ammonium acetate to 2.5M; add 2.5 volumes absolute ethanol. Store at -80C, 30 min
-Spin 15-20 minutes in microfuge at top speed.
-Pour off supernatant and wash pellets with 1 ml 70% EtOH. Mix by inversion, spin again 5 min.
-pour off supernatant and dry pellets.
-resuspend thoroughly the RNA/ probe pellet in 30 ul formamide hybridization buffer: 40mM PIPES pH 6.4, 400mM NaCl, 1mM EDTA, 80% deionized formamide
-cover hybridization with 50ul paraffin oil to prevent evaporation
-Incubate hybridization at 90 degrees in dry-bath incubator for 15 minutes to denature probe.
-carry hot block to water bath set at proper hybridizing temperature (generally about 37-52 degrees, determined empirically, but depending on GC content and length of hybrid that can form). 37 degrees often works fine.
-quickly move tube from 90 degree hot block to 37 degree water bath.
-hybridize 2 hours to overnight.
-chill tubes on ice, or if maintaining stringency is important, pop open tubes in water bath and add S1 digestion mix.
-add to each tube 270 ul of ice-cold S1 digestion mix, containing about 125 units S1 nuclease/ml (S1 is added, just before use, to a volume of S1 digestion buffer appropriate for the number of transcription reactions).
S1 digestion buffer
5% glycerol, 1mM Zinc Sulfate, 30mM NaOAc (sodium acetate), pH 4.5, 50mM NaCl
-spin 5 seconds in microfuge to get S1 digestion mix through the paraffin oil layer.
-vortex briefly. Repeat spin to get thin layer of oil at top again.
-incubate 30min in 37 degree Celsius water bath.
-stop reactions by removing 280 ul from the bottom of the tube (avoid paraffin oil) to a fresh tube containing:
10ul of 10%SDS
5ul of 0.5M EDTA (the EDTA chelates the Zinc, stopping enzymatic activity)
-vortex briefly to mix.
-add 30ul 7.5M Ammonium acetate and vortex
-add 1ml cold (kept in freezer) absolute ethanol to precipitate DNA/RNA hybrid and vortex. store -80, 30'.
-Spin 15 -20 minutes at top speed in microfuge (room temperature is OK, 4 degrees won't hurt).
-carefully remove supernatant (pellets may not be visible) and discard in radioactive waste container.
-add 1ml cold (kept in freezer) 70% Ethanol, mix by inversion twice, and spin 5 minutes.
-carefully remove supernatant and dry pellets under vacuum (or in 65 degree water bath with tube caps open).
-resuspend pellets in 6-8 ul of formamide-dye (sequencing gel loading buffer), amended to contain NaOH to chew up RNA in the RNA/DNA hybrid:
Formamide-dye Loading Buffer: 90% deionized formamide (deionized with 0.25g mixed bed resin per 10ml formamide) 10mM NaOH, 1mM EDTA, 0.1% (1mg/ml)Bromophenol blue, 0.1% (1mg/ml) Xylene cyanol
-boil 3 min., chill on ice. Load on 6 or 8% Urea-PAGE (sequencing) gel beside end-labeled pBR322 Hpa II digested (or other) molecular weight markers.