Home

About

Advanced Search

Browse by Discipline

Scientific Societies

E-print Alerts

Add E-prints

E-print Network
FAQHELPSITE MAPCONTACT US


  Advanced Search  

 
Probe Synthesis Random Primed Labeling of DNA fragments with NEBlot kit
 

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 l) at 100C
for 5 minutes.
2. Plunge tube into ice and chill for 5 minutes. Spin down DNA solution.
3. Set up reaction: 2 l dATP
2 l dGTP
2 l dTTP
5 l 10X buffer + primers
5 l [32P]dCTP (Alpha label)
1 l Klenow
Flick with finger to mix, and spin down reaction volume.
4. Incubate at 37C for 60 minutes.
5. Add 1 l 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 -20C for 1-3 days. Use radioactive shielding.
5'-End Labeling of Oligonucleotides:
1. Dilute oligonucleotide probe to ~100 ng/l in TE or ddH2O. For an average 19-mer, 1 l = ~17

  

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

 

Collections: Biology and Medicine