Radioactive labelling of Probes
Oligo Labelling Buffer (OLB) : Consists of a 2:5:3 ratio of solutions A:B:C
Solution A: 15 ml 20mM dATP, 15 ml 20 mM dCTP, 15 ml 20mM dGTP, 15 ml 20mM dTTP, 18 ml b-mercaptoethanol, 937 ml solution O. Solution O: 1.25mM Tris.Cl pH 8.0, 0.125M MgCl 2
Solution B: 2M Hepes pH 6.6
Solution C: 25 ng/ ml random hexadeoxyribonucleotides.
50ng of DNA probe is denatured in 13ml of dH20 by boiling for ten minutes. It is immediately transferred to ice in order to retain the denaturised state.
5 ml of OLB and 1 ml BSA (10mg/ml) are added on ice and mixed by pipette. 5 ml of radioactive [ a -32P ] dCTP ( ~ 1.85 MBq) with a specific activity of 111TBq/mM and a concentration of 370 MBq/ml (NEN) is then added to the DNA probe. Next 1 ml Klenow fragment (1U/ml, Promega) is added. This reaction is then incubated for 2Hrs at 37°C.
In order to remove unincorporated nucleotides from the probe, it is passed over a sephadex G50 column (Amersham Pharmacia), and collected in an Eppendorf tube. A further 50ml of TE is added to the eluted probe. The radioactive labelled probe is then denatured by boiling the tube for 5 minutes in water, and transferred immediately to ice.