Reagents

  • 25-50ng of target DNA in no more than 25µl TE buffer.

  • Ready.To.Go DNA Labelling Kit (-dCTP), Pharmacia, 27-9251-01

  • ProbeQuant G-50 Micro Columns, Pharmacia, 27-5335-01

  • ddH2O

  • 5ml scintillation fluid in vial.

 

Protocol

Probe synthesis

1. Reconstitute the contents of the Reaction Mix tube by adding 20µl 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 °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)                  <25µl

         [a-32P]dCTP (3000 Ci/mmol)                5µl          (50µCi)

         ddH2O                                    to total of 50µl

4. Mix by gently pipetting up and down several times. Bubbles may be removed by a pulse centrifugation.

5. Incubate at 37 °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.

 

Probe purification

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 50µl 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 �20 °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)1/2 . For example with a rotor radius of 73mm, the appropriate speed would be 3000rpm.

 

Probe quantification

12. Resuspend 1µl of purified probe in 50µl 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/µg. Yields are usually >106 cpm/µl.

 

Probe qualification

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 °C for 1 hour.

19. Expose phosphoscreen.

Note: PCR products should give one sharp band.

Department of Microbiology
School of Medicine
University of Washington
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