1. Prepare reaction cocktail on ice:
| Quantity | Substance | Source |
|---|---|---|
| 2.5 uL | 0.5 mM 3dNTP mix (no dATP) | (NEB #N0446S) |
| 2.5 uL | 10X Klenow Buffer | supplied with Klenow order |
| 5.0 uL | 3000 Ci/mmol α32P dATP | your radioactive supplier |
| 1.0 uL | Klenow (3 to 8 units) | (NEB #M0210S) |
| 11 uL /rxn | ||
2. Combine your probe (30 to 100 ng) with random 9mers (1 to 5 ug, from NEB #S1254S) in total volume of 14 uL.
Denature by boiling (make sure cap is secure) for 2 to 3 minutes (using boil beads is easiest), spin down, then place on ice.
| Quantity | Substance | Source |
|---|---|---|
| 2-3 uL | DNA probe | |
| 5 uL | 1 ug/uL 9mer | (NEB #S1254S |
| 6-7 uL | water | |
| 14 uL /rxn | ||
Use a screw-cap eppendorf or an eppendorf clamp to prevent an explosion of radioactive material while boiling in the denaturing step!
3. Add 11 uL of the reaction mix from Step 1 to the denatured DNA (final volume 25 uL) and incubate 37°C for 30 minutes.
4. Stop reaction by adding:
| Quantity | Substance | Source |
|---|---|---|
| 1 uL | 0.5 M EDTA | |
| 3 uL | 10 mg/ml tRNA | (Ambion #7119) |
| 100 uL | TE buffer |
5. Remove unicorporated labelled nucleotides with ProbeQuant G-50 Microcolumns.
GE Healthcare (formerly Amersham) # 27-5335-01 for a box of 50 columns.