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Kansas State University

 

PREPARATION AND PURIFIATION OF BIOTIN-LABELED PROBES FOR IN SITU HYBRIDIZATION (ISH)


Suggested chemicals: Nick Translation Kit, Enzo Diagnostics Cat. No. 42803.

 

Biotin labeling of plasmid or genomic DNA

  1. Prepare the following hybridization mixture.
  2. dNTP (including biotin-11-dUTP
    5 無
    plasmid or genomic DNA
    1 痢
    DNA polymerase I
    5 無
    Dnase I
    X µL
    Deionized or distilled water
    Y µL
    Total reaction volume (use H2O to adjust the total volume to 20 µL)
    50 µL
  3. Incubate the reaction tube in a water bath at 14-15°C for 2 h.
  4. Stop the reaction by adding 5 痞 stop buffer.

The most critical part of the labeling, if the DNA is clean, is the size of the final nick-translation product. The best ISH results are obtained when the size of the biotin-labeled probe is around 300-600 bp. To determine the size of the probe, denature 15 無 of the nick-translation product in boiling water for 4 min, cool on ice immediately, and electrophorese the sample on a minigel with a DNA size marker. The size of the nick-translation product can be adjusted by adding a different amount of DNase I to the reaction solution. Random-primer labeling also can be used for biotin labeling, however the size of the probe is relatively easier to control by nick translation.

Purification of the biotin-labeled probe

  1. 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 to the top with Sephadex G-50 using a sterilized pasteur pipette.
  3. Place the Sephadex-filled syringe into a 15-mL Corex tube and centrifuge at 1,500 rpm for 4 min to pack down the Sephadex.
  4. Repeat the previous two steps until the packed Sephadex column has a total volume of about 0.9 mL.
  5. Wash the colum twice with 55 µL TE by spinning at 1,500 rpm for 4 min each.
  6. Place a sterilized 1.5-mL tube on the bottom of the 15-mL Corex tube and insert the packed and washed syringe into the Corex tube in such a way that the tip of the syringe fits into the 1.5-mL tube. Load the nick translation product (55 µL with stop buffer) and spin at 1,500 rpm for 4 min. The probe is collected in the 1.5-mL tube.

The final volume of probe should be close to 55 無. Usually, 5 無 of such a biotin-labeled probe (1 痢/55 無 = 18 ng/無) is enough for one slide, and the probe can be used to process 100 slides. The size of the probe can be checked by electrophoresis on a minigel, either before or after the spin-colum purification. The probe can be stored in a freezer for several years.