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.
- QIAquick PCR Purification kit
- Spin column tubes
- 1.5mL epis
- 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.
- 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.
- Place a QIAquick spin column in a provided 2 ml collection tube.
- To bind DNA, apply the sample to the QIAquick column and let sit for 1 minute. Centrifuge for 30 seconds at 9,000rpm.
- 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.
- 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.
- 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.
- 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.
- 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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