Knight:Micropure EZ and Microcon purification

Overview

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

• Micropure-EZ filter
• Microcon filter
• Tabletop centrifuge
• Sterile H₂O

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. The remaining steps are for a YM-30 column. Other columns may require different spin speeds and times.
2. Add entire restriction digest volume to Micropure-EZ filter.
3. Spin 8 mins in table top centrifuge at 10,000 g. Spin at room temperature.
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. Spin at room temperature.
8. Discard flow-through.
9. Add 200 μL H₂O to Microcon column.
10. Spin 8 mins in table top centrifuge at 10,000 g. Spin at room temperature.
    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.

Notes

• For a 57 bp fragment, use the above protocol except use a YM-10 microcon columns and increase the spin times from 8 mins to 30 mins.
• If you find a large volume is being retained in the column, this is usually an indication that you loaded a lot of material in the column. It is not necessary to add 20μL of water prior to the elution spin.
• The spin steps should be carried out at room temperature rather than 4°C otherwise longer spin times are needed for complete spin through of the sample.