Probe Labelling

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Biotin/digoxigenin-labelling of Plasmid or Genomic DNA

1.     Prepare following reaction solution:

        10X nick-translation buffer                                 5 µl

        dNTP solution                                                    2.5 µl        

        dUTP solution (biotin or digoxigenin)               2.5 µl                                                   

        Plasmid or Genomic DNA                                  1 µg

        DNA Polymerase I                                             12 units

        DNase I                                          

        H₂O                                                                 

        Reaction volume (total)                                    50 µl

2.     Use H₂O to adjust the total volume to 50 µl.  Carefully mix after each step of adding solutions and spin down at the end.

3.     Incubate reaction tube in water bath at 14-15˚C for 2 hrs.

4.     Stop the reaction by adding 5 µl stop buffer (200 mM EDTA).

Notes:

a.     10X nick-translation buffer: 0.5 M Tris HCl pH 7.5, 50 mM MgCl₂.  dNTP solution: containing 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM dCTP.  dUTP solution: 0.166 mM dUTP (biotin or digoxigenin-congugated) and 0.333 mM dTTP . 

b.     The most critical part for the labelling (if the DNA is clean) is the size of the final nick translation product.  The best in situ hybridization results will be obtained when the size of the biotin-labeled probe is around 200-600 bp.

c.     For determining the size of the probe, denature 15 µl of the nick translation product in boiling water for 4 min and cool down on ice immediately.  Run the sample on a mini-gel with a DNA size marker.

d.     The size of the nick translation product can be adjusted by adding different amount of DNase I in the reaction solution.

e.     Random priming technique can also be used for labelling.  Since the size of probe is relatively easier to control by nick translation, we prefer to use the nick translation technique.

f.     We suggest you to buy a nick-translation kit if you don’t have enough experience.  Several companies sell such kits which include the biotin-dUTP or digoxigenin-dUTP.

II.  Probe Purification

1.     Start 20 min before the end of the nick translation incubation. Plug the bottom of a 1 ml tuberculin syringe with siliconized glass wool.

2.     Fill the syringe with Sephadex G-50 up to the top using a sterilized pasteur pipette.

3.     Place the Sephadex-filled syringe into a 15 ml Corex tube.

4.     Centrifuge at 1500 rpm for 4 min to pack down the sephadex. 

5.     Repeat step 2 to 4 so that the packed sephadex column has a total volume of about 0.9 ml.

6.     Wash the column twice with 55 µl TE by spin at 1500 rpm for 4 min each.

7.     Place a sterilized 1.5 ml tube on the bottom of the 15 ml Corex tube, insert the packed and washed syringe into the corex tube such that the tip of the syringe is fitted into the 1.5 ml tube.  Load the nick translation product (55 µl, with stop buffer) and spin at 1500 rpm for 4 min.

8.     The probe DNA will be collected in the 1.5 ml tube.

Notes:

a.     If you did not make any mistakes, the final volume should be close to 55 µl.  Usually 0.5 µl of such biotin-labelled probe (1 µg/55 µl = 18 ng/µl) is enough for each slide for in situ hybridization.  Thus you can use the probe for at least 100 slides.

b.     You can check the size of the probe (by running a mini-gel) either before or after the spin-column purification.

c.     The probe can be stored in a freezer for several years.  You may store it at 4°C if you use it regularly.

d.     The spin-column method can be replaced by other techniques for probe purification.  You can use an easier ethanol precipitation method.