Single-stranded synthetic oligos are ideal for DNA probes. Oligos 20 - 60 nt long are sufficient and work well.
Phosphatase Treatment
Optional: Treat probe DNA (synthetic oligo) with Antarctic Phosphatase to dephosphorylate the 5’-ends. Dephosphorylating probe prior to end labeling increases specific activity. This step is usually not necessary for a synthetic oligo unless you ordered it with a 5´-phosphate or have phosphorylated it enzymatically.
1. Combine:
§ 1 μL of 10x Antarctic Phosphatase reaction buffer
§ 1 μg of probe DNA
§ 1 μL (5 units) of Antarctic Phosphatase
§ H2O to 10 μL total volume
2. Incubate15 min at 37°C (for 5’ overhang)
3. Heat inactivate 5 min at 65°C
4. Can scale reaction up and store a stock of dephosphorylated probe at –20°C. Label aliquots as needed.
γ-ATP label 5’ ends with Polynucleotide Kinase