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MOLECULAR BIOLOGY: WORKING WITH DNA

NUCLEIC ACID PROBES

RADIOLABELING OF OLIGONUCLEOTIDES

Radiolabeling of Oligonucleotides Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

Procedure Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

1. Add the following to a microcentrifuge tube: 1 μl of 50 ng/μl oligonucleotide, dephosphorylated at the 5' termini 0.5 μl of Denaturing Buffer 3.5 μl of ddH2O 2. Incubate the mixture for 5 minutes at 70°C. 3. Quick chill the reaction by placing the tube on ice for 5 minutes and briefly spin to remove condensation from lid. 4. Add the following components sequentially to the tube: 1.25 μl of 10X Kinase Buffer 0.5 μl of 100 mM DTT 5 μl of γ-[32P]-dATP 0.75 μl of T4 Polynucleotide kinase. 5. Incubate the reaction for 60 minutes at 37°C. 6. After completion of the incubation, add: 36 μl of TE 50 μl of 4 M Ammonium Acetate 1 μl of 10 mg/ml E. coli tRNA 200 μl of Ethanol 7. Mix the reaction by vortexing and centrifuge at top speed in a microcentrifuge for 10 minutes. 8. Carefully remove the supernatant and discard it. Resuspend the labeled oligonucleotide pellet in 100 μl of TE. 9. Count 1 μl by scintillation counting (see Protocol on scintillation counting of radioactive samples).

Solutions Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

4 M Ammonium Acetate T4 Polynucleotide Kinase    5 Units/μl γ-[32P]-dATP (CAUTION See Hint #3)    γ-[32P]-dATP 3000 Ci/mmol, 10 uCi/μl 100mM DTT 50% Glycerol    50% (v/v) Glycerol Kinase Buffer (10X)    500 mM Tris-HCl, pH 9.5 100 mM MgCl2 Denaturing Buffer (See Hint #2)    10mM EDTA pH 8.0 10 mM Spermidine 200 mM Tris-HCl, pH 9.5 TE    10 mM Tris-HCl, pH 8.0 1 mM EDTA. Oligonucleotide    50 ng/μl in TE (See Hint #1) 10 mg/ml E. coli tRNA

BioReagents and Chemicals Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

EDTA γ-[32P]-dATP Ammonium Acetate Spermidine DTT Polynucleotide Kinase, T4 Magnesium Chloride tRNA, E. coli Glycerol Tris-HCl Oligonucleotide Ethanol

Protocol Hints Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

1. Ammonium ions strongly inhibit the kinase activity of T4 Polynucleotide Kinase. Do not dissolve the oligonucleotide in buffers containing ammonium salts or precipitate the oligo from such buffers. 2. Spermidine stimulates the incorporation of γ-[32P]-dATP into the probe. 3. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.