Section on Neural Gene Expression

PREPARATION OF OLIGONUCLEOTIDE PROBES FOR HYBRIDIZATION HISTOCHEMISTRY

Terminal Deoxynucleotidyl Transferase Labeling of 3' Ends:

We label our oligonucleotides, generally about 48 bases long, using terminal deoxynucleotidyl transferase and alpha-35S-dATP. The resultant probes have specific activities (>10,000Ci/mmol) of about 10 times the specific activity of the alpha-35S-dATP.

Materials

Terminal deoxynucleotidyl transferase (TdT)
5X tailing buffer (500mM potassium cacodylate (pH 7.2), 10mM CoCl2, 1.0mM DTT)
alpha-35S-dATP (>1000Ci/mmol)
TE (10mM Tris - HCl, 1mM EDTA, pH 7.6)
4M NaCl
25 µg/µl yeast tRNA
Ethanol
Dithiothreitol (DTT)

Preparation of probe

  1. On ice, mix 10 µl 5x tailing buffer, 0.1µM oligonucleotide, 1µM 35S-dATP, 70-100 units of TdT and H2O to 50 µl.

    The 35S-dATP should not come in a solution containing EDTA or similar chelator that will interfere with the tailing buffer (a color change indicates that this has happened). alpha-32P-dATP is easily substituted for alpha-35S-dATP at the same concentration in this reaction to make probes for northern analysis. For a 48 base oligonucleotide, 0.1µM is about 1.56ng/µl.

  2. Incubate approximately 2-5 min. at 37° C to add 10-15 bases.

  3. The enzyme lot may be examined to assess activity and number of nucleotides added.

  4. Add 375 µl TE, 25 µl 4M NaCl, 50 µg tRNA and 1 ml ethanol. Mix thoroughly, place on wet ice for 10 min. and spin in a microcentrifuge at 13,000 rpm for 10 min.

  5. Remove the supernatant and rinse the pellet with 1 ml of 70% ethanol. Remove the ethanol and add 50 µl TE and 1 µl 5M DTT. Count 1 µl of radiolabeled probe in a scintillation counter. May be stored at 4° C.

  6. Expect about 500,000 dpm per µl. At this point, one can estimate the specific activity based on the total amounts of alpha-35S-dATP and oligonucleotide added and the amount of radioactivity recovered, conservatively assuming 50% recovery.

  7. For digoxigenin-labeled probes, use 1 µl of a 250µM digoxigenin-dUTP/1mM dNTP (or dATP) mix (separate components; digoxigenin-dUTP from Boehringer Mannheim). Incubate for 2 hrs. and dissolve the precipitated probe in 50 µl TE . Use 3-10 µl/100 µl hybridization solution.

T4 Kinase Labeling of 5' Ends:

  1. Reaction mix:

    • 3µl 10x kinase buffer (10x=0.7M Tris-HCl, pH7.6; 0.1M MgCl2; 50mM DTT). The BRL Cycle Sequencing buffer (5X; Y01329) has phosphatase inhibitors.

    • oligonucleotide at 1.5µM final

    • gamma-35S-ATP (NEN NEG-039H, ~1300Ci/mmol) or gamma-32P-ATP (NEG-002H, ~2000Ci/mmol) at 2µM final

    • T4 kinase (10U; BRL 8004SA)

    • Water to 30µl
  2. Incubate for 2hrs. at 37°C for gamma-35S-ATP or 30min for gamma-32P-ATP.

  3. Add 400µl TE, 22.5µl 4M NaCl, 1µl glycogen, and 450µl phenol/chloroform. Extract.

  4. Extract supernatant with 420µl chloroform.

  5. Add 1ml ethanol and precipitate on wet ice for 10min. Spin 10min. Rinse pellet with 70% ethanol. Redissolve pellet in 50µl TE with 100mM DTT (for 35S). Store at 4°C.



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