Random primer generation of [32P]-labelled DNA probes

DNA probes are prepared using a modification of the method of Feinberg and Vogelstein, (1983).

You will need:

Nuclease-free BSA [a-32P]-dCTP (Amersham or DuPont) Klenow DNA Polymerase (New England Biolabs or Pharmacia) 500mM EDTA, pH 8 dNTP solutions (separate solutions of 100mM dATP, dGTP, dCTP and dTTP in TE, pH 7.0 Solution O (1.25M Tris.Cl, 125mM MgCl2 , pH 8.0) Solution A (1ml solution O, 18ul b-mercaptoethanol, 5ul of each dNTP solution Solution B (2M HEPES, pH 6.6) Solution C (Random hexanucleotides at 90 OD260 nm/ml) Sterile, nano-pure water

1) Digest plasmid DNA with the appropriate restriction enzyme,fractionate electrophoretically, and purify by the NaI/glass method (see Gene-Klene Protocol). Resuspend in sterile,distilled H2 O at approximately 2ng/ul.

2) Boil DNA at 100°C or 10 minutes.

3) Snap chill in an ice/water bath and hold on ice prior to use.

4) Carry out labelling reactions at room temperature by the addition, to a sterile Eppendorf tube, of the following reagents in the stated order:-

5ul OLB buffer (see below) 1ul 10mg/ml nuclease free BSA 17.5ul (25ng) DNA fragment 370KBq [a-32P]-dCTP (1ul of a 370KBq/ul stock) 0.5ul Klenow fragment of E. coli DNA polymerase (0.5-1 unit) OLB buffer is made by mixing solutions A, B and C in the ratio 10: 25: 15, respectively.

5) Allow reactions to continue for at least 5 hours.

6) Terminate reactions by the addition of 1ul 0.5M EDTA, 74ul sterile, distilled H2 O and 5 minutes incubation at 100°C.

The specific activity and the percentage incorporation of 32P-dCTP into the probe should be determined by TCA precipitation of 1ml probe and scintillation counting.