Setting Up Nick Translation Reaction
- Add the following to a microcentrifuge or PCR tube on ice and make up to final volume of 20 µl.
| BAC DNA: |
2 µl (100 ng) |
| Biotin dUTP (40 µM): |
2 µl |
| 10x Nick Translation buffer: |
2 µl |
| dNTP (100 µM each): |
4 µl |
| [α-32P] dCTP: |
1 µl |
| DNase I/DNA Polymerase mixture: |
2 µl |
| water: |
7 µl |
| total: |
20 µl |
- Incubate for 90 min (or shorter) at 15 °C.
Isotope is included as a tracer to confirm that biotinylation reaction has proceeded efficiently.
- Stop the reaction by adding 1 µl 0.5 M EDTA pH 8.0 or by heating to 65°C for 10 min.
- Purify the biotinylated DNA by column chromatography through a micro bio-spin P-30 column (Bio-Rad).
[α-32P]dCTP (3000Ci/mmol) (Amersham or Perken Elmer); DNase I/DNA Polymerase (Roche); dNTP (Invitrogen); Biotin dUTP (Enzo Biochem).
Streptavidin Bead Binding Buffer:
10 mM Tris-HCl (pH 7.5)
1 mM EDTA (pH 8)
1 M NaCl
2X Hybridization Buffer:
1.5 M NaCl
40 mM Sodium phosphate buffer (pH7.2)
10 mM EDTA (pH 8.0)
10x Denhardt's
0.2% SDS