Qiagen (R) DNeasy (R) DNA Purification Protocol

A. Summary

The Qiagen DNeasy kit includes instructions for grinding and extracting DNA, as well as purifying it. The bench-guide protocol below only describes how to use the Qiagen kit to purify DNA that has already been extracted, and is thus a modified version of the original Qiagen protocol. You can consult the DNeasy Plant Handbook for more information

B. Materials & Reagents

1. Materials

  • a. Pipettes and tips
  • b. DNeasy 0.5 mL Mini spin Purification columns
  • c. DNeasy 2 mL Collection tubes
  • d. 1.5 mL Snap-top Microcentrifuge tubes
  • e. Centrifuge, with 1.5 mL tube adaptors, capable of 14,000 rpm
  • f. -20C Freezer
  • g. Vortexer
  • h. Water Bath/Incubator

2. Reagents

  • a. RNAse
  • b. Buffer AP3
  • c. Buffer AW (from DNeasy kit)
  • d. Buffer AE (from DNeasy kit)
  • e. Deionized, distilled water (ddH2O)

C. Procedure

(step # from DNeasy Plant Handbook in parentheses)

1. (step 7) Combine the following and incubate at 37C for 30 minutes:

  • a. 40 - 50 uL DNA
  • b. 1 uL RNase (optional)
  • c. 200 uL AE Buffer or TE buffer or water

2. (step 13) Add AP3 Buffer at 1.5 times the volume of the above mixture, i.e. 375 uL, and pipette-mix the solution thoroughly.

3. (step 14) Transfer the solution from step 2 to a white Mini spin column (using the same tip as in step 2), and place the Mini spin column in a 2 mL Collection tube.

  • a. Centrifuge the Mini spin column, while in the Collection tube, for 1 minute at 8000 rpm.
  • b. Discard flow-through, and place discarded Collection tubes in a beaker to cleaned and reused.

4. (step 16) Place Mini spin column in new 2 mL Collection tube.

  • a. Add 500 uL of Buffer AW to the Mini spin column.
  • b. Centrifuge for 1 minute at 8000 rpm.
  • c. Discard the flow-through, but KEEP the Collection tube for the next step.

5. (step 17) Add 500 uL of Buffer AW to the Mini spin column.

  • a. Centrifuge for 2 minutes at 14,000 rpm, to remove any residual ethanol from column.
  • b. Discard flow-through and place discarded Collection tubes in a beaker to be cleaned and reused.

6. (step 18) Transfer Mini spin column to a new snap-top 1.5 mL microcentrifuge tube.

  • a. Warm Buffer AE at 65C for 5 - 10 minutes.
  • b. Add 50 uL of warm Buffer AE to Mini spin column.
  • c. Incubate for 5 minutes at room temperature.
  • d. Centrifuge Mini spin column, in a new snap-top tube, for 1 minute at 8000 rpm.
  • e. Keep elution, and label appropriately ("1st elution").

7. (step 19) Repeat step 7 above for a "2nd elution", which will likely be at a lower concentration than the "1st elution" but will still be useful for various applications.