9 Radioactive Labeling of DNA and RNA Probes
115
In all protocols described in this chapter, 33 P can be substituted for 32 P in
applications where isotope emissions that are less energetic than 32 P are
desired ( e.g., cycle sequencing or in situ hybridization) (McLaughlin and
Margolskee 1993; Evans and Read 1992).
Materials
Synthetic oligodeoxyribonucleotides should have a free 5' hydroxyl group Substrate
(i.e., trityl group removed if synthesized using phosphoramidite chemistry).
Dephosphorylate (see Chap. 2) or use the exchange kinase reaction (see below) if a 5' phosphate group is present. Double-stranded DNA may also be
labeled, but protruding 5' ends are labeled much more efficiently than blunt
or recessed 5' ends.
• Bacteriophage T4 polynucleotide kinase (10 units/Jll).
Other reagents
• y[ 32 P]ATP (3000 Ci/mmol, 10 mCi/ml)
• 1 Ox reaction buffer is:
- 0.5 M Tris-Cl (pH 7.6)
- 0.1 M MgCh
- 50 mM dithiothreitol
- 1 mM spermidine
- 1 mM EDTA (pH 8.0)
• 1.5 ml microcentrifuge tube (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)
Procedure
1. Combine in order in a sterile 1.5 ml microcentrifuge tube on ice:
- 50 pmol synthetic oligonucleotide
- 2.5 J.1l10x reaction buffer
- 10 J.!l y[ 32 P]ATP (33 pmol)
Supplies and
equipment
Labeling 5' ends
with T4
115
In all protocols described in this chapter, 33 P can be substituted for 32 P in
applications where isotope emissions that are less energetic than 32 P are
desired ( e.g., cycle sequencing or in situ hybridization) (McLaughlin and
Margolskee 1993; Evans and Read 1992).
Materials
Synthetic oligodeoxyribonucleotides should have a free 5' hydroxyl group Substrate
(i.e., trityl group removed if synthesized using phosphoramidite chemistry).
Dephosphorylate (see Chap. 2) or use the exchange kinase reaction (see below) if a 5' phosphate group is present. Double-stranded DNA may also be
labeled, but protruding 5' ends are labeled much more efficiently than blunt
or recessed 5' ends.
• Bacteriophage T4 polynucleotide kinase (10 units/Jll).
Other reagents
• y[ 32 P]ATP (3000 Ci/mmol, 10 mCi/ml)
• 1 Ox reaction buffer is:
- 0.5 M Tris-Cl (pH 7.6)
- 0.1 M MgCh
- 50 mM dithiothreitol
- 1 mM spermidine
- 1 mM EDTA (pH 8.0)
• 1.5 ml microcentrifuge tube (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)
Procedure
1. Combine in order in a sterile 1.5 ml microcentrifuge tube on ice:
- 50 pmol synthetic oligonucleotide
- 2.5 J.1l10x reaction buffer
- 10 J.!l y[ 32 P]ATP (33 pmol)
Supplies and
equipment
Labeling 5' ends
with T4