120
PETER IGARASHI
- 1 Jll 0.5 mM dGTP (25 J.!M final)
- 1 Jll 0.5 mM dTTP (25 J.!M final)
- 5 J.!l a[ 32 P]dCTP (0.83 J.!M final)
- 1 Jll Klenow fragment of E. coli DNA polymerase I (2 units)
4. Incubate at 37°C for 30 minutes.
5. While the reaction is incubating, prepare a Sephadex G-50 spin column
by centrifugation at 2000x g for 2 min (optional).
6. Stop the reaction by adding 2J.1l of0.2 M EDTA (pH 8.0). Load the reaction mixture onto the spin column and centrifuge at 2000x g for 4 min
( optional).
7. Count 1J.1l of column eluatein a liquid scintillation counter. Prior to use
in hybridization experiments, the probe should be denatured by heating
at 100°C for 5 minutes, then placed immediately on ice for 5 min.
Comments
• The DNA template should be linear.
• Random hexamers are available commercially or may be synthesized on
an automated DNA synthesizer using equimolar concentrations of all
four bases at each position.
• The Klenow fragment ofDNA polymerase I is sensitive to denaturation.
A void vortexing the reaction mixture.
• Incubating Ionger than 30 min at 37°C may result in reduced specific
activity of the probe. Alternatively, incubations may be performed at
room temperature for 2 hours.
• 32 P-labeled probe that is prepared using this method is susceptible to
radiolysis and should be used promptly.
• DNA fragments can also be labeled directly in low-melting-point agarose
gels (e.g., NuSieve) (Feinberg and Vogelstein 1984). Gelslices containing
template DNA are excised and diluted with three times their weight of
water. The slices are melted at 95°C for 7 min, then placed in a 37°C water
bath. 9 Jll of the melted gel solution is then removed and labeled as described above (starting with step 3) except that the reaction mixture is
kept at 37°C to prevent gelling.
PETER IGARASHI
- 1 Jll 0.5 mM dGTP (25 J.!M final)
- 1 Jll 0.5 mM dTTP (25 J.!M final)
- 5 J.!l a[ 32 P]dCTP (0.83 J.!M final)
- 1 Jll Klenow fragment of E. coli DNA polymerase I (2 units)
4. Incubate at 37°C for 30 minutes.
5. While the reaction is incubating, prepare a Sephadex G-50 spin column
by centrifugation at 2000x g for 2 min (optional).
6. Stop the reaction by adding 2J.1l of0.2 M EDTA (pH 8.0). Load the reaction mixture onto the spin column and centrifuge at 2000x g for 4 min
( optional).
7. Count 1J.1l of column eluatein a liquid scintillation counter. Prior to use
in hybridization experiments, the probe should be denatured by heating
at 100°C for 5 minutes, then placed immediately on ice for 5 min.
Comments
• The DNA template should be linear.
• Random hexamers are available commercially or may be synthesized on
an automated DNA synthesizer using equimolar concentrations of all
four bases at each position.
• The Klenow fragment ofDNA polymerase I is sensitive to denaturation.
A void vortexing the reaction mixture.
• Incubating Ionger than 30 min at 37°C may result in reduced specific
activity of the probe. Alternatively, incubations may be performed at
room temperature for 2 hours.
• 32 P-labeled probe that is prepared using this method is susceptible to
radiolysis and should be used promptly.
• DNA fragments can also be labeled directly in low-melting-point agarose
gels (e.g., NuSieve) (Feinberg and Vogelstein 1984). Gelslices containing
template DNA are excised and diluted with three times their weight of
water. The slices are melted at 95°C for 7 min, then placed in a 37°C water
bath. 9 Jll of the melted gel solution is then removed and labeled as described above (starting with step 3) except that the reaction mixture is
kept at 37°C to prevent gelling.