Summary: 45
Probe Synthesis
Random Primed Labeling of DNA fragments with NEBlot kit:
1. Denature 25 ng of isolated DNA fragment in nuclease-free ddH2O (final volume = 33 µl) at 100°C
for 5 minutes.
2. Plunge tube into ice and chill for 5 minutes. Spin down DNA solution.
3. Set up reaction: 2 µl dATP
2 µl dGTP
2 µl dTTP
5 µl 10X buffer + primers
5 µl [32P]dCTP (Alpha label)
1 µl Klenow
Flick with finger to mix, and spin down reaction volume.
4. Incubate at 37°C for 60 minutes.
5. Add 1 µl 0.5 M EDTA, pH 8, and mix. Place reaction on ice.
6. Separate unincorporated counts on spin column. Estimate incorporation with monitor (should be
33-67%).
7. Use probe immediately, or store at -20°C for 1-3 days. Use radioactive shielding.
5'-End Labeling of Oligonucleotides:
1. Dilute oligonucleotide probe to ~100 ng/µl in TE or ddH2O. For an average 19-mer, 1 µl = ~17

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

Collections: Biology and Medicine