54
TAKEHIKO KOJI
size ofless than 200-400 bp (as assessed by ethidium bromide
staining of agarose-gel after electrophoresis of probe DNA) is
recommended. Since the usual size of oligo-DNA is less than
100 bases, no significant problem is encountered.
Procedures for dot-blot hybridization with T-T dimerized
oligo-DNA probe
1. Cut nitrocellulose filters and immerse them in 20 X SSC (1 X
SSC=0.15 M NaCl/ 0.015 M Na citrate (pH 7.0» for 30 mins,
and air-dry on Parafilm.
2. Apply sense oligo-DNA (boiled for 5 mins and chilled
quickly at 4 0c) to the filters at 1 pg to 10 ng/2 Ill/spot.
3. After air-drying, bake the filters for 2 hrs at 80°C.
4. Prehybridize for 2 hrs at 37°C in a medium (boiled for 7 mins
and chilled quickly at 4 0c) consisting of 10 mM Tris/HCI
buffer (pH 7.4) containing 0.6 M NaCI, 1 mMEDTA, 1 XDenhardt's solution, 500 /lg/ml yeast tRNA, 250 /lg/ml salmon
testis DNA and 40% formamide.
5. Hybridize at 37°C overnight in a medium (boiled for 7 mins
and chilled quickly at 4 0c) consisting of 10 mM Tris/HCI
buffer (pH 7.4) containing 0.6 M NaCI, 1 mMEDTA, 1 XDenhardt's solution, 250 /lg/ml yeast tRNA, 125 /lg/ml salmon
testis DNA, 10% dextran sulfate, 40% formamide and 1-2
/lg/ml T-T dimerized antisense oligo-DNA.
6. Wash with 2 X SSC (30 mins, 2 times), 1 X SSC (30 mins, 2
times), 0.5 X SSC (at room temperature to 45°C; 15 mins,
once) and PBS (15 mins, once), successively.
7. Immerse for 1 hr in PBS containing 5% BSA, 500 /lg/ml normal mouse IgG, 100 /lg/ml yeast tRNA and 100 /lg/ml salmon
testis DNA.
8. React for 1 hr with HRP-Iabeled monoclonal anti-(T -T dimer) antibody dissolved in PBS containing 5% BSA, 100
/lg/ml yeast tRNA and 100 /lg/ml salmon testis DNA.
9. Wash 5 times with 0.075% Brij in PBS for 15 mins each time.
TAKEHIKO KOJI
size ofless than 200-400 bp (as assessed by ethidium bromide
staining of agarose-gel after electrophoresis of probe DNA) is
recommended. Since the usual size of oligo-DNA is less than
100 bases, no significant problem is encountered.
Procedures for dot-blot hybridization with T-T dimerized
oligo-DNA probe
1. Cut nitrocellulose filters and immerse them in 20 X SSC (1 X
SSC=0.15 M NaCl/ 0.015 M Na citrate (pH 7.0» for 30 mins,
and air-dry on Parafilm.
2. Apply sense oligo-DNA (boiled for 5 mins and chilled
quickly at 4 0c) to the filters at 1 pg to 10 ng/2 Ill/spot.
3. After air-drying, bake the filters for 2 hrs at 80°C.
4. Prehybridize for 2 hrs at 37°C in a medium (boiled for 7 mins
and chilled quickly at 4 0c) consisting of 10 mM Tris/HCI
buffer (pH 7.4) containing 0.6 M NaCI, 1 mMEDTA, 1 XDenhardt's solution, 500 /lg/ml yeast tRNA, 250 /lg/ml salmon
testis DNA and 40% formamide.
5. Hybridize at 37°C overnight in a medium (boiled for 7 mins
and chilled quickly at 4 0c) consisting of 10 mM Tris/HCI
buffer (pH 7.4) containing 0.6 M NaCI, 1 mMEDTA, 1 XDenhardt's solution, 250 /lg/ml yeast tRNA, 125 /lg/ml salmon
testis DNA, 10% dextran sulfate, 40% formamide and 1-2
/lg/ml T-T dimerized antisense oligo-DNA.
6. Wash with 2 X SSC (30 mins, 2 times), 1 X SSC (30 mins, 2
times), 0.5 X SSC (at room temperature to 45°C; 15 mins,
once) and PBS (15 mins, once), successively.
7. Immerse for 1 hr in PBS containing 5% BSA, 500 /lg/ml normal mouse IgG, 100 /lg/ml yeast tRNA and 100 /lg/ml salmon
testis DNA.
8. React for 1 hr with HRP-Iabeled monoclonal anti-(T -T dimer) antibody dissolved in PBS containing 5% BSA, 100
/lg/ml yeast tRNA and 100 /lg/ml salmon testis DNA.
9. Wash 5 times with 0.075% Brij in PBS for 15 mins each time.