Choice of Label:
NEBlot® Kit can be used with the labeled nucleotide of your choice. Listed below are guidelines for the use of some common nucleotide triphosphates used in labeling reactions.
Random Priming Reaction:
Allow system components to thaw on ice. Klenow enzyme, however, should be kept at –20°C except when being used.
Standard 50 μl reaction using 32P dCTP
For a standard 50 μl reactoin, total volume of DNA and label should be 38 μl.
- In a microcentrifuge tube:
Dissolve 25 ng of template DNA in nuclease free H2O (1-33 μl).
For the control reaction, add 1μl of control DNA to 32 μl of H2O.
- Denature in boiling H2O bath for 5 minutes (see manual Figure 1, page 7).
- Quickly place in ice for 5 minutes.
- Centrifuge briefly in the cold.
- Add the following reagents to your DNA in the order listed.
• 5 μl Octadeoxyribonucleotides in 10X Labeling Buffer
• 6 μl dNTP mixture (2 μl of dATP, dTTP, and dGTP).
• 5 μl α 32P dCTP (3,000 ci/mmol, 50 μCi)
• 1 μl DNA Polymerase I – Klenow Fragment (3’ → 5’ exo-) (5 units)
- Incubate at 37oC for 1 hour (see manual Figure 1, page 4 for other labels).
Incubation time for DNA/Agarose 6 hours for 32P [ ]
- Terminate reaction by adding 5 μl of 0.3 M EDTA (pH 8.0).
We recommend purification of probes to reduce non-specific background and limit exposure of lab personnel to high levels of radioactivity during hybridization experiments.