Knight:Micropure EZ and Microcon purification

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This protocol was used to purify a DNA fragment about 80 bp in length. The fragment encoded a promoter and was generated via restriction digest. This protocol will remove enzymes and salts. It also removes species that have lower molecular weight than what the Microcon filter retains. Any DNA or RNA with higher molecular weight than what the column retains will remain in your sample. For 3A assembly, the presence of the vector fragment does not matter cause incorrect products are selected against.

Materials

Procedure

  1. Read literature that comes with the Micropure-EZ and Microcon filter carefully. It contains a lot of useful information. It also describes how to assemble the Micropure-EZ filter on top of the Microcon filter in a vial. For an 80 bp fragment, I used the YM-30 column successfully.
  2. Add entire restriction digest volume to Micropure-EZ filter.
  3. Spin 8 mins in table top centrifuge at 10,000 g. Room-temperature is fine.
  4. Discard Micropure-EZ filter but keep Microcon filter. (The Micropure-EZ filter trapped the enzymes.)
  5. Discard flow-through.
  6. Add 200 μL H2O to Microcon column.
  7. Spin 8 mins in table top centrifuge at 10,000 g. Room-temperature is fine.
  8. Discard flow-through.
  9. Add 200 μL H2O to Microcon column.
  10. Spin 8 mins in table top centrifuge at 10,000 g. Room-temperature is fine.
    The above two wash steps remove salts from the restriction digest.
  11. Discard flow-through.
  12. Move Microcon filter to new eppendorf tube.
  13. Add 20 μL H2O to Microcon column.
  14. Invert column in tube.
    The column won't be very stable but it should remain in place for the next step.
  15. Spin for 3 mins at 1000 g.
  16. Discard Microcon column. The liquid in the tube should contain your purified sample.
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