Radiolabeling DNA Probe for Northerns
25-50ng of target DNA in no more than 25Âµl TE buffer.
Ready.To.Go DNA Labelling Kit (-dCTP), Pharmacia, 27-9251-01
ProbeQuant G-50 Micro Columns, Pharmacia, 27-5335-01
5ml scintillation fluid in vial.
1. Reconstitute the contents of the Reaction Mix tube by adding 20Âµl ddH2O. DO NOT MIX. Let sit on ice for 5-60 minutes.
2. Denature 25-50ng DNA by heating for 2-3 minutes at 95-100 Â°C. Immediately place on ice for 2 minutes, then centrifuge briefly.
3. Add the following to the reconstituted Reaction Mix tube:
Denatured DNA (25-50ng) 25Âµl
[a-32P]dCTP (3000 Ci/mmol) 5Âµl (50ÂµCi)
ddH2O to total of 50Âµl
4. Mix by gently pipetting up and down several times. Bubbles may be removed by a pulse centrifugation.
5. Incubate at 37 Â°C for 5-15 minutes. (Difficult templates may require up to 30 minutes.)
Note: The Reaction Mix contains dATP, dGTP, dTTP, FPLC-pure Klenow fragment (4-8 units) and random oligonucleotides, primarily 9-mers.
6. Resuspend the resin in spin column by vortexing.
7. Loosen the cap one-fourth and snap off the bottom closure.
8. Place the column in a 1.5ml screw-cap tube.
9. Pre-spin the column for 1 minute at 735 x g (3500rpm).
10. Place the column in a new 1.5ml tube and slowly apply 50Âµl of the sample to the top-center of the resin without disturbing the resin-bed.
11. Spin the column at 735 x g for 2 minutes. The purified probe is collected in the bottom of the support tube. Cap the tube. Store at ï¿½20 Â°C until use.
Note: For a force of 735 x g the appropriate speed can be calculated from the following formula: rpm = (1000) (657/r)½ . For example with a rotor radius of 73mm, the appropriate speed would be 3000rpm.
12. Resuspend 1Âµl of purified probe in 50Âµl ddH2O.
13. Transfer mixture to a vial with 5ml scintillation fluid.
14. Count the samples (as user 7, T-wing).
Note: this protocol should label 25-50ng of DNA to 1 x 109 dpm/Âµg. Yields are usually 106 cpm/Âµl.
15. Run 20,000 cpm probe on a 1% Agarose gel.
16. Place gel on 4 pieces of Whatman paper.
17. Cover just the gel with piece of Saran Wrap.
18. Dry down gel at 65 Â°C for 1 hour.
19. Expose phosphoscreen.
Note: PCR products should give one sharp band.