Stripping Southern Blots for Reuse with Another Probe see Note

1. Fill a beaker or glass dish about half to two-thirds full with 0.1X SSC/1% SDS using a heated magnetic stirrer and heat the buffer to boiling.

2. Add the blot to be stripped and reduce heat to a low, steady boil. Cover with a glass plate, or other cover.

3. Boil for about 30 min with gentle stirring. Check the reduction of radioactive signal strength of the blot with a Geiger counter. If necessary, boil again in fresh buffer.

4. Expose the stripped blot wet, wrapped in plastic, to film under the same conditions that it will be exposed to for the next hybridization to check for any residual probe signal left.

5. Before hybridizing the stripped blot, agitate in 0.4 NNaOH for 1 min and then in 0.2 M Tris-HCl, pH 8.0/1X SSC for 1 min to make sure the DNA fixed on the membrane is denatured.

4. Notes

1. Make sure all buffers, rotors, beakers, tubes, and so on are prechilled to 0°C to 4°C.

2. Break up the pellet well with a 1-mL plastic pipet before Dounce homogenization.

3. Nuclei are very fragile in this buffer and will lyse upon harsh treatment such as high g-force or Dounce homogenization.

4. This will give an effective Ca2+ concentration of 1 mM.

5. All washes should be discarded into the 32P liquid radioactive waste.

6. There may be a loss of up to 50% of the DNA fixed to the blot after stripping.


This work was supported by NIH grant GM62857, NIGMS to A.S.


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