You will need:
[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 OD260nm/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 H2O 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 H2O 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.
Dr. Simon Dawson,
Department of Biochemistry,
University of Nottingham,
The Medical School ,
Clifton Boulevard ,
Tel: +44 (0)115 9249924 ex.44787,
FAX: +44 (0)115 9422225,
Click here to return to the Submission Form or here to go to the Methods Finder.