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EasyPrep PCR Cleaning Protocol

(Centrifuge method)

  1. Column preparation: add 100 µl Column Buffer to the membrane of the DNA column, incubate for 2-3 min, centrifuge at 11,000 rpm for 30-60s. Discard the flowthrough and put the column back to the collection tube.
  2. Add 5 volume Buffer PB to the PCR reaction and mix well. (i.e. add 250 µl PB to 50 µl PCR reaction). Briefly spin the tube to collect any drops from the inside wall and tube lid. It’s not necessary to remove mineral oil.

  3. Transfer up to 750 µl of the mixture to a DNA mini column with a collection tube. Centrifuge at 11,000 rpm for 1 min. Discard the flowthrough and put the column back to the collection tube. Repeat this step to process if there is remaining mixture.

  4. Add 750 µl DNA Wash Buffer to the column and centrifuge at 11,000 rpm for 1 min at room temperature. Discard the flow through. Ensure that ethanol has been added to DNA Wash Buffer before use.

  5. Centrifuge the empty column at 11,000 rpm for 1 min to remove the residual ethanol. It is critical to remove residual ethanol for optimal elution. Residual ethanol may interfere subsequent applications.

  6. Place the column in a clean 1.5 ml micocentrifuge tube. Add 20-35 µl Elution Buffer or H2O (pH7.5-8.5) to the column. Incubate at room temperature for 1 min. Centrifuge at 11,000 rpm for 1 min to elute the DNA. If it’s a large fragment (≥10 kb), pre-warm the elution buffer to 65oC will increase the yield.

  7. Optional: Re-apply the eluate to the column, elute one more time. This will increase DNA yield.

(Vacuum method)

  1. Prepare the vacuum manifold according to manufacturer’s instructions. Attach the spin column to the manifold. Add 100 µl Column Buffer to the membrane of the DNA column, incubate for 2-3 min, Switch on vacuum source to draw the solution through the columns completely, and then switch off vacuum source.
  2. Add 5 volume Buffer PB to the PCR reaction and mix well. (i.e. add 250 µl PB to 50 µl PCR reaction). Briefly spin the tube to collect any drops from the inside wall and tube lid.  It’s not necessary to remove mineral oil.

  3. Prepare the vacuum manifold according to manufacturer’s instructions. Attach the spin column to the manifold.

  4. Transfer up to 750 µl DNA/Buffer GC mixture to a DNA mini column. Switch on vacuum source to draw the solution through the columns completely, and then switch off vacuum source.

  5. Add 750 µl DNA Wash Buffer to the column. Switch on vacuum source to draw the solution through the columns completely and then switch off vacuum source. Ensure that ethanol has been added to DNA Wash Buffer before use.

  6. Transfer the DNA columns to 2 ml collection tubes. Centrifuge at 11,000 rpm for 1 min. It is critical to remove residual ethanol for optimal elution. Residual ethanol may interfere subsequent applications.

  7. Place the column in a clean 1.5 ml micocentrifuge tube. Add 20-35 µl Elution Buffer or H2O (pH7.5-8.5) to the column. Incubate at room temperature for 1 min. Centrifuge at 11,000 rpm for 1 min to elute the DNA. If it’s a large fragment (≥10 kb), pre-warm the elution buffer to 65oC will increase the yield.

  8. Optional: Re-apply the eluate to the column, elute one more time. This will increase DNA yield.

 

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