9 Radioactive Labeling of DNA and RNA Probes
119
Materials
Double-stranded DNA (50-200 ng). Minimallength has not been deter- Substrate
mined, but we have labeled 135-bp fragments successfully. Plasmid DNA
should be linearized prior to use. U sually restriction fragments or PCR products are isolated on agarase gels and purified with silica resins ( Qiaex,
GeneClean, Prep-a-Gene, etc.) prior to use. Plasmid DNA can also be
used but non-specific hybridization will be higher.
• Klenow fragment of E. coli DNA polymerase I (2 unit/J..Ll).
Other reagents
• a[ 32 P]dCTP (3000 Ci/mmol, 10 mCi/ml)
• lOx reaction buffer: 0.5 M Tris-Cl (pH 7.2), 0.1 M MgC12, 1 mM dithioerythritol, 2 mg/ml BSA, 2.5 mg/ml random hexamers
• 0.5 mM each of dATP, dGTP, dTTP
• 1.5 ml microcentrifuge tubes (autoclaved)
• Table top centrifuge
• Microcentrifuge
• 37°C water bath
• 100°C heat block
• Liquid scintillation counter
• Sephadex G-50 spin columns (Boehringer-Mannheim) or alternative
method for separating probe from unincorporated radionucleotide ( optional)
Procerlure
1. Combine in a sterile 1.5 ml microcentrifuge tube:
- 10-200 ng template DNA (in 1-9 J..Ll water)
- Sterile water to 9 J..Ll
2. Heat to 100°C for 5 minutes then place immediatelyon ice. Spin brieflyin
a microcentrifuge.
3. Add in order on ice:
- 2 J..Ll 1 Ox reaction buffer
- 1 J..Ll 0.5 mM dATP (25 J..LM final)
Supplies and
eqUipment