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PETERIGARASHI
- 1.5 fll T4 polynucleotide kinase (15 units)
- to 25 fll sterile water
2. Incubate at 37°C for 30 minutes.
3. While the reaction is incubating, prepare a Sephadex G-25 spin column
by centrifugation at 2000x g for 2 minutes (optional).
4. Stop the reaction by adding 2fll of0.5 M EDTA or heating to 68°C for 10
min. Load the reaction mixture on to the spin column and centrifuge at
2000x g for 4 min (optional). Count 1 fll of column eluate in a liquid
scintillation counter.
Note: lt is important to avoid contamination of substrates with ammonium
ions which inhibit polynucleotide kinase (e.g., avoid prior ethanol precipitation with ammonium chloride).
Subprotocol 2
End-Labeling Using the Exchange Kinase Reaction
Materials
Substrate Synthetic oligodeoxyribonucleotide or double-stranded DNA containing a
5' phosphate group.
Other reagents • Bacteriophage T4 polynucleotide kinase {10 units/f.!l).
• y[ 32 P]ATP {3000 Ci/mmol, 10 mCi/ml)
• 5 mM ADP
• 10x reaction buffer: 0.5 M imidazole-Cl (pH 6.6), 0.1 M MgClz, 50 mM
DTT, 1 mM spermidine, 1 mM EDT A
Procedure
1. Combine in a sterile 1.5 ml microcentrifuge tube on ice:
- 50 pmol synthetic oligonucleotide
- 2.5 fll 1 Ox reaction buffer
- 1.5 fll ADP {300 f.!M final)
- 10 fll y[ 32 P]ATP (33 pmol)
PETERIGARASHI
- 1.5 fll T4 polynucleotide kinase (15 units)
- to 25 fll sterile water
2. Incubate at 37°C for 30 minutes.
3. While the reaction is incubating, prepare a Sephadex G-25 spin column
by centrifugation at 2000x g for 2 minutes (optional).
4. Stop the reaction by adding 2fll of0.5 M EDTA or heating to 68°C for 10
min. Load the reaction mixture on to the spin column and centrifuge at
2000x g for 4 min (optional). Count 1 fll of column eluate in a liquid
scintillation counter.
Note: lt is important to avoid contamination of substrates with ammonium
ions which inhibit polynucleotide kinase (e.g., avoid prior ethanol precipitation with ammonium chloride).
Subprotocol 2
End-Labeling Using the Exchange Kinase Reaction
Materials
Substrate Synthetic oligodeoxyribonucleotide or double-stranded DNA containing a
5' phosphate group.
Other reagents • Bacteriophage T4 polynucleotide kinase {10 units/f.!l).
• y[ 32 P]ATP {3000 Ci/mmol, 10 mCi/ml)
• 5 mM ADP
• 10x reaction buffer: 0.5 M imidazole-Cl (pH 6.6), 0.1 M MgClz, 50 mM
DTT, 1 mM spermidine, 1 mM EDT A
Procedure
1. Combine in a sterile 1.5 ml microcentrifuge tube on ice:
- 50 pmol synthetic oligonucleotide
- 2.5 fll 1 Ox reaction buffer
- 1.5 fll ADP {300 f.!M final)
- 10 fll y[ 32 P]ATP (33 pmol)