Column Purification of Restriction Enzyme Digest

 

Equipment & Consumables:

  • Sterile Micropipette and tips

  • Full PPE: Gloves, Lab Coat and Glasses

  • Buffer PB

  • Buffer PE

  • Buffer EB

  • Appropriate grade spin columns for your fragment sizes and capture tubes

  • Microcentrifuge tubes

  • Kimwipes + Oven (or a heat block)

  • Your Restriction Enzyme Digest samples.

Protocol:

  1. Put on gloves + safety glasses. PB contains guanidine which is very toxic.

  2. Figure out what volume of sample you have. If this is less than 100 µl, then make it up to 100 µl with TE. The volume you now have will be called “1 volume”. Add 5 volumes of buffer PB to the DNA solution. e.g. if you have a 100 µl restriction digest, add 500 µl PB.

  3. Place a silica-based spin column into its 2 ml catch tube (if it isn’t already set up that way). Load up to 750 µl of the DNA-PB mixture onto the column. Spin at ~10,000 g for 30 sec. Discard the flow-through into culture waste.

  4. If you still have more DNA-PB mixture left, repeat the previous step until all of the mixture has been put thru the column. The columns will hold a total of ~10 µg DNA, which is a lot!

  5. Add 750 ml of buffer PE to the column, spin ~10,000 g for 30 sec, discard flow-through.  

  6. Repeat step 4.

  7. Spin again for 30 sec to remove all traces of PE from the column. Discard both the flow-through and catch tube, and transfer the spin column onto a clean Kimwipe. Leave the column lid open. Transfer Kimwipe to 60°C oven, and allow to dry for 10 min.

    • Or leave the columns in clean capture tubes with lids open in a heat block at the same temperature.

  8. Transfer spin column to a sterile 1.5 ml Eppi tube, and add 15-50 µl* of EB buffer to the centre of the spin column – ie on the membrane, not the walls of tube. Allow to sit for 2 min. Spin at ~10,000 g for 1 min, retain Eppi tube with DNA solution in EB, discard spin column. 

    • Optional: Add all of eluted volume back to the column, allow to sit for 2 mins and spin again at max speed for 1-2 minutes. Can increase yield

  9. Move to Ligation Protocol if you now wish to stick these two parts together and/or Agarose Electrophoresis Protocol for immediate analysis of the digest.

    • *Usually we want high concentration rather than high yield, so use 20 µl. If max. yield is important, or if you have lots of DNA, use 50 µl. Note that you lose approx 5 ml EB during the procedure.  

Acknowledgements: