QIA Quick PCR Purification

Summary

This protocol is designed to purify single or double-stranded DNA fragments from PCR and other enzymatic reactions. Revised from QIAquick Spin Handbook, pg. 19.

Equipment

  • Microcentrifuge

Materials

  • QIAquick PCR Purification kit
  • Spin column tubes
  • 1.5mL epis

Protocol

  1. Add 5 volumes of Buffer PBI to 1 volume of the PCR sample (e.g. 500 microliters buffer to 100 microliters PCR sample) and mix. Be careful- this stuff is super toxic. Wipe up spills immediately and don't allow contact with your skin. It not necessary to remove mineral oil or kerosene.
  2. Check that color of mixture is yellow (similar to Buffer PBI without the PCR sample). IF color is orange or violet, add 10 microliters of 3 M sodium acetate, pH 5.0, and mix. The color will turn yellow.
  3. Place a QIAquick spin column in a provided 2 ml collection tube.
  4. To bind DNA, apply the sample to the QIAquick column and let sit for 1 minute. Centrifuge for 30 seconds at 9,000rpm.
  5. Discard flow through into buffer waste container. Place the QIAquick column back in the same tube. Collection tubes are re-used to reduce plastic waste.
  6. To wash, add 0.75 ml Buffer PE to the QIAquick column and let sit for 1 minute (longer if your downstream application is salt sensitive). Centrifuge for 30 sec at 9000rpm.
  7. Discard flow through into buffer waste container and place the QIAquick column back in the same tube. Centrifuge column for an additional 1 minute at 13,000rpm. IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow through is discarded before this additional centrifugation.
  8. Check insides and bottom of filter tube to make sure there are no remaining drops of ethanol. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
  9. To elute DNA, add 30 microliters hot Milli-Q water (heated to 65 degrees Celsius) to the center of the QIAquick membrane and let stand for 1 minute. Centrifuge the column for 1 minute at 13,000rpm. IMPORTANT: Make sure pH of water is between 7.0-8.5 to achieve maximum elution efficiency. Store DNA at -20 degrees Celsius as DNA may degrade in absence of buffering agent.

Original Protocol from QIAquick Spin Handbook

Protocol Updated by: H. Lindsey Date: 12 Sept 12

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