Preparation of DNA & RNA Probes
Protocol 1. Standard DNA random primers labeling protocol.
1. Denature 25-100ng DNA
dissolved in 20ul of ddH2O in a microcentrifuge tube by heating for 5 minutes in a boiling water bath, then immediately cool on ice.
2.
Add the following reagents on ice and mixt gently.
1 ul 500uM dATP
1 ul 500uM dGTP
1 ul 500uM dTTP
10ul 5x random primers solution
5
ul (about 50 uCi) alpha-32P-dCTP
ddH2O to a total volume of 49 ul.
3. Add 1 ul Klenow enzyme. Mix gently but thoroughly. Centrifuge
305 seconds.
4. Incubate at 37 centigrade for 10-15 minutes.
5. Add 5 ul stop buffer (0.5 M EDTA).
6. This labeled
DNA fragments can be used for hybridization.
Protocol 2. Synthesis of single strand DNA probe
Protocol 3. Radiolabeling
probes by PCR
Protocol 4. Synthesis of cDNA probes from mRNA