Biotechnology and Bioinformatics (1+1)

Method

1. Digest DNA with an appropriate RE. Purify by gel electrophoresis or ethanol precipitation. Resuspend in TE buffer.

2. Dossolve 25ng of template DNA in nuclease free water (1-34 m l)

3. Denature in boiling water for 5 min and chill on ice for 5 min. Centrifuge briefly.

4. Add the following to the DNA

5.0 m l 10X Klenow fragment buffer

6.0 m l dNTP mix (2 m l of 0.5mM dTTP, dCTP, dGTP)

4.0 m l 3000Ci/mmol [ a ^-32 P]dATP (40 m Ci)

1.0 m l Klenow fragment (5U)

5. Incubate for 1hr at 37⁰C.

6. Stop reaction with 1 m l of 0.5M EDTA.

7. Separate labeled DNA from unincorporated radioactive precursors by column chromatography.

8. Remove 1 m l aliquot and determine ³²P labeling by Scintillation counter. Specific activity should be 10⁹ cpm/ m g DNA.