Linearize template plasmid with proper restriction enzyme.

Extract with PCA
EtOH ppt., wash and dry.
Dissolve to about 0.5 µg/µl.

Regular riboprobe:
10 µl:
1 µl 10xT7 buffer
1 µl 50 mM DTT
1 µl 5 mM ATP, GTP, CTP, 0.1 mM UTP
0.5 µl lineraized plasmid (≈0.5 µg/µl)
2.5 µl a 32 P-UTP (10 mCi/ml)
0.5 µl RNasin
0.5 µl SP6 or T7 polymerase
(Note: Omission of cold UTP can enhance the signal from the existing probe if alpha-32P is not fresh.)

37C, 1 h

Centrifuge

37C, 1 h

Add 40 µl ddH2O
Place a Sephadex G50 column in an eppendorf tube w/o lid and spin at 3,000 rpm, 1 min. Transfer spin column to new tube.
Add Probe mix to center of spin column.
Spin at 3,000 rpm, 2 min.
Transfer eluate (containing probe minus nucleotides) to new tube.
Check on a geiger counter that probe is hot.
Count 1 µl in scintillation counter.
Use 10 6 -10 7 cpm per Northern blot (should be around 10-15 µl).

Strip-EZ probe (Ambion):
10 µl:
1 µl 10xT7 buffer
1 µl 50 mM DTT
1 µl 5 mM ATP, GTP, 0.1 mM UTP
0.25 µl 2mM modified CTP (comes with kit)
0.5 µl lineraized plasmid (≈0.25 µg/µl)
2.5 µl a 32 P-UTP (10 mCi/ml)
0.5 µl RNasin
0.5 µl SP6 or T7 polymerase

37C, 1 h.

40 µl ddH2O

Place a Sephadex G50 column in an eppendorf tube w/o lid and spin at 3,000 rpm, 1 min. Transfer spin column to new tube.
Add Probe mix to center of spin column.
Spin at 3,000 rpm, 2 min.
Transfer eluate (containing probe minus nucleotides) to new tube.
Check on a geiger counter that probe is hot.
Count 1 µl in scintillation counter.
Use 10 6 -10 7 cpm per Northern blot (should be around 10-15 µl).

Buffers
10xT7 buffer:
400 mM Tris-HCl pH 8.0
60 mM MgCl 2

KT 050413