The David H. Koch Institute for Integrative Cancer Research at MITThe David H. Koch Institute for Integrative Cancer Research at MIT

Massachusetts Institute of Technology

National Cancer Institute Cancer Center

Science + Engineering... Conquering Cancer Together

How to Make Hot Probes for Your Southern

1. Prepare reaction cocktail on ice:

QuantitySubstanceSource
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. USE A SCEW-CAP EPPENDORF or an eppendorf clamp to prevent an explosion of radioactive material while boiling in the Denaturing step!!!

QuantitySubstanceSource
2-3 uL DNA probe  
5 uL 1 ug/uL 9mer (NEB #S1254S)
6-7 uL water  
14 uL /rxn

Denature by boiling (make sure cap is secure) for 2 to 3 minutes (using boil beads is easiest), spin down, then place on ice.

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:

QuantitySubstanceSource
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).