9 Radioactive Labeling of DNA and RNA Probes
117
- 1.5 J..Ll T4 polynucleotide kinase (15 units)
- to 25 J..Ll sterile water
2. Incubate at 37°C for 30 min.
3. Stop the reaction by adding 2J..Ll of0.5 M EDTA or heating to 68°C for 10
min. Remove unincorporated nucleotides by centrifugation through a
Sephadex G-25 spin column (2000x g for 4 min) (optional). Count 1
J..Ll of column eluate in a liquid scintillation counter.
Note: The exchange kinase reaction is much less efficient than the "forward"
reaction but is satisfactory for labeling molecular weight standards, for example.
Subprotocol 3
labeling 3' Ends with Terminal Transferase (Forward Reaction)
For principle considerations see Subprotocol 1, Description.
Materials
Synthetic oligodeoxyribonucleotide, double-stranded DNA (protruding 3' Substrate
ends are preferred), or single-stranded DNA containing a free 3' hydroxyl
group.
• Terminaltransferase (15 units/J..Ll).
Other reagents
• a[3 2 P]dATP (3000 Ci/mmol, 10 mCi/ml)
• 5x reaction buffer: 0.5 M potassium cacodylate (pH 7.2), 10 mM CoClz,
1 mM DTT
• 1.5 ml microcentrifuge tubes (autoclaved)
• Table top centrifuge with swinging bucket rotor ( optional)
• 37°C water bath
• Liquid scintillation counter
• Sephadex G-25 spin columns (Boehringer-Mannheim) or alternative
method for separating probe from unincorporated radionucleotide ( optional)
Supplies and
equipment
117
- 1.5 J..Ll T4 polynucleotide kinase (15 units)
- to 25 J..Ll sterile water
2. Incubate at 37°C for 30 min.
3. Stop the reaction by adding 2J..Ll of0.5 M EDTA or heating to 68°C for 10
min. Remove unincorporated nucleotides by centrifugation through a
Sephadex G-25 spin column (2000x g for 4 min) (optional). Count 1
J..Ll of column eluate in a liquid scintillation counter.
Note: The exchange kinase reaction is much less efficient than the "forward"
reaction but is satisfactory for labeling molecular weight standards, for example.
Subprotocol 3
labeling 3' Ends with Terminal Transferase (Forward Reaction)
For principle considerations see Subprotocol 1, Description.
Materials
Synthetic oligodeoxyribonucleotide, double-stranded DNA (protruding 3' Substrate
ends are preferred), or single-stranded DNA containing a free 3' hydroxyl
group.
• Terminaltransferase (15 units/J..Ll).
Other reagents
• a[3 2 P]dATP (3000 Ci/mmol, 10 mCi/ml)
• 5x reaction buffer: 0.5 M potassium cacodylate (pH 7.2), 10 mM CoClz,
1 mM DTT
• 1.5 ml microcentrifuge tubes (autoclaved)
• Table top centrifuge with swinging bucket rotor ( optional)
• 37°C water bath
• Liquid scintillation counter
• Sephadex G-25 spin columns (Boehringer-Mannheim) or alternative
method for separating probe from unincorporated radionucleotide ( optional)
Supplies and
equipment