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BURT G. FEUERSTEIN et al.
- Buffer C (PCR labeling reaction)
- 8.3 I-tl 600l-tM dNTPs (100l-tM final concentration)
- 7.0 1-t11Ol-tM DOP primer
- 6.0 I-tl MgCh (3 mM final concentration)
- 5.0 I-tl lOx PCR buffer (1.5 mM MgCh)
- 0.5 I-tl Taq polymerase (1.5 units)
- 15.7l-t1 H 2 0
low stringency
(Sequenase)
reaction
High stringency
(Taq) reaction
PCR labeling
reaction
1. Dispense 4 I-tl Buffer A and 1 I-tl DNA sample to the appropriate tube,
and overlay with 3 drops of mineral oil (total reaction volume 5 I-tl).
2. Denature at 96°C for 10 min.
3. Subject to 5 cycles of 30°C for 5 min, 37°C for 2 min, and 96°C for 2 min.
Add 0.65 units ofSequenase at each 30° step. A thermostable sequenase
now available (Amersham) may allow one to omit the latter additions
of enzyme.
1. Immediately after the Sequenase reactions have been completed, add
45 JlI of Buffer B (total reaction volume 50 JlI).
2. Denature at 96°C for 5 min.
3. Subject to 35 cycles of 94°C for 1 min, 56°C for 2 min, and noc for
2 min with autoextension of 1 s/cycle.
4. Final extension at noc for 5 min.
5. Soak samples at 4°C until retrieval.
6. Dispense 42.5 I-tl Buffer C, 2.5 I-tl digoxygenin-dUTP (50 I-tM final concentration) (Roche), and 5 I-tl DOP-PCR product into each tube (total
reaction volume 50 I-tl). Overlay each sample with mineral oil.
7. Denature at 95°C forIO min.
8. Subject to 25 cycles of at 94°C for 70 s, 56°C for 70 s, and noc for 3 min
with autoextension of 1 s/cycle.
9. Final extension at noc for 10 min.
10. Soak samples at 4°C until retrieval.
11. Reference DNA is amplified from 50 ng normal DNA and labeled as
described above except that biotin (Boehringer/Mannheim) or fluorescein isothiocyanate (FITC)-dUTP (Du Pont) is substituted for DIGdUTP.