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DNA Probe Purification

I. Spin method

  1. Determine the volume of the labeling reaction and transfer to a clean microtube, add 5 volume Buffer PI. Mix the solution well by vortexing. Briefly spin the tube to collect any drops from the inside wall and tube lid. Note: Radio-isotopic labeling reactions yield sub microgram of DNA due to the limited nucleotide concentration used. In this case, a carrier such as yeast tRNA should be added to Buffer PI to a final concentration of 2-5 µg/ml to ensure the binding of the DNA to the matrix.
  2. Transfer up to 700 µl of the probe reaction/ Buffer PI solution to a spin column with a collection tube. Centrifuge at 13,000 x g for 1 min at room temperature. Discard the flowthrough and insert the column back to the collection tube. Repeat this step to process all remaining solution.
  3. Add 500 µl DNA Wash Buffer to the column and centrifuge at 13,000 x g for 1 min at room temperature. Discard the flowthrough. Ensure that ethanol has been added to DNA Wash Buffer as instructed before use.
  4. Repeat step 3.
  5. Centrifuge the empty column at top speed for 1 min to remove the residual ethanol in the column. This is critical for DNA yield.
  6. Optional: Spin the empty column with the collection for 1 min.
  7. Place the column into a clean 1.5 ml micocentrifuge tube and add 30-50 µl Elution Buffer or ddH2O to the center of the column. Incubate at room temperature for 1 min. Centrifuge at 13,000 x g for 1 min to elute the DNA.

II.  Vacuum/Spin Protocol

  1. Determine the volume of the labeling reaction and transfer to a clean microtube, add 5 volume Buffer PI. Mix the solution well by vortexing. Briefly spin the tube to collect any drops from the inside wall and tube lid.
  2. Prepare the vacuum manifold according to manufacturer’s instructions. Attach the column to the manifold.
  3. Load the probe reaction/ Buffer PI solution to the column. Turn on the vacuum to let the solution pass through the column.
  4.  Wash the column by adding 500 µl DNA Wash Buffer.
  5. Repeat step 4.
  6. Assemble the column in a collection tube and spin at top speed for 2 min.
  7. Assemble the column to a clean 1.5 ml tube, add 3050 µl Elution Buffer or ddH2O, and incubate at room temperature for 1 min. Centrifuge the column at 13,000 x g to elute DNA.

Note: 

  • This represents approximately 80-90% of the bound DNA. An optional second elution will yield any residual DNA.
  • Probes greater than 500 bp in length can routinely be purified at > 60% recovery with greater than 95% removal of free nucleotides. Fragments ranging from 50 bp to 500 bp in length normally give yield of 30 – 60% of the DNA.

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