9 Radioactive Labeling of DNA and RNA Probes
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Procedure
1. Add in order in a 1.5 ml sterile microcentrifuge tube (at room temperature):
- 5 J..Ll 5x transcription buffer
1 J..Lg Template DNA
1.2 J..Ll10 mM rATP (480 J..LM final)
1.2 J..Ll 10 mM rCTP (480 J..LM final)
1.2 J..Ll10 mM rGTP (480 J..LM final)
1 J..Ll 1 mM UTP ( 40 J..LM final)
1 J..Ll 0.75 M dithiothreitol
1 J..Ll RNAse Block II or 0.5 J..Ll RNAsin
5 J..Ll [a- 32 P]UTP (2.5 J..LM final) or a- 35 S-UTP (2 J..LM final)
to 25 J..Ll DEPC-treated water
1 J..Ll T3, T7 or SP6 RNA polymerase (10 units)
2. Incubate at 37-40°C for 60 min.
3. If gel purification will not be done:
a. Add 1 J..Ll of ribonuclease-free deoxyribonuclease, and incubate 15 min
at 37°C.
b. Increase the volume to 100 J..Ll and extract once with 100 J..Ll ofphenol:
chloroform:IAA (25:24:1).
c. Extract once with 100 J..Ll of chloroform:IAA (24:1}.
d. Add 0.5 volume of7.5 M ammonium acetate and 2 volumes of ethanol.
Place on dry ice for 30 minutes.
e. Centrifuge at 10,000x g for at least 5 min ( drain supernatant and monitor with Geiger counter).
f. Resuspend the pellet in 100 J..Ll1 M ammonium acetate and repeat steps
d and e.
g. Dry the pelletunder vacuum (Speed-Vac) and resuspend in DEPCtreated water. Count 1 J..Ll in a liquid scintillation counter.
4. If gel purification will be done:
a. Omit treatment with RQ1 RNAse-free DNAse
b. Prepare a sequencing gel ( 5% polyacrylamide/6M urea)
c. Bring riboprobe volume to 100 J..Ll with DEPC-treated water and extract
once with 100 J..Ll of phenol:chloroform:IAA (25:24:1}. Extract once with
100 J..Ll of chloroform:IAA (24:1).
d. Add 0.5 volumes of7.5 M ammonium acetate. Add 2 volumes of ethanol, and place on dry ice for 30 min.
e. Centrifuge at 10,000xg for atleast 5 min (drain supernatant and moni-