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