Making Hot Probes For Southerns

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.