Biochemical Techniques and Instrumentation

[32P]-Labelling of Probe

1. Add 25 ng of DNA to a total volume of 10μl of H₂O in a microfuge tube.
2. Boil x 10' and place on ice. Spin down 10 sec in a microfuge
3. Immediately add:
   3 ul of dATP, dGTP, dTTp mixture - Thaw rapidly and
   2 ul solution 6 (reaction mixture) - keep on ice
   5 ul [a32P] dCTP (3000 Ci/mM)-must have reference date within 1 wk of labelling
   1 ul Klenow enzyme (5 U/ml)
4. Incubate 37°C x 45'
5. Add 2μl 500 mM EDTA and place at 65°C for 10' to stop reaction.
6. Prepare spin column:
   1. Place G-50 spin column (Boehringer) and collection tube in 15 ml polystyrene tube
   2. Spin 3000 RPM x 2'
   3. Discard collection tube and replace it with another in polystyrene tube
7. Add 2μl yeast tRNA (10 mg/ml in H₂O) and 50μl STE to hexa priming mixture and mix.
8. Load hexapriming mix onto spin column
9. Spin 3000 RPM x 4-5'
10. Discard column and place effluent in eppendorf tube.
11. Count 1μl of effluent to determine the specific activity of the probe. The specific activity should be at least 1 x 109 DPM/μg DNA
12. Boil probe x 10' and place on dry ice prior to use.

Solutions:

1. tRNA: yeast tRNA (Sigma) in H₂O at 10 mg/ml. Store 4°C.
2. STE: 10 mM Tris (pH 7.5) Store R.T.
   1 mM EDTA
   100 mM NaCl