IGEM:Yale/2010/Protocols/PCR purification

Standard PCR Purification Protocols

Unless stated otherwise, all PCR purification was accomplished using a QIAquick PCR Purification Kit. These protocols are taken from the QIAquick Spin Handbook.

Vacuum Manifold Protocol

1. Add 5 volumes of Puffer PB to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene.
2. Prepare the vacuum manifold and QIAquick columns.
3. To bind DNA, load the samples into the QIAquick columns by decanting or pipetting, and apply vacuum. After the sample shave passed through the column, switch off the vacuum source.
4. To wash, add 0.75 mL of Buffer PE to each QIAquick column and apply vacuum.
5. Transfer each QIAquick column into a clean 1.5 mL microcentrifuge tube.
6. To dilute DNA, add 40 uL Buffer EB or water to the center of the QIAquick membrane, let stand for one minute, then centrifuge the column for 1 minute. Repeat with 15 mL to elute remaining DNA.

Microcentrifuge Protocol

1. Add 5 volumes of Puffer PB to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene.
2. Place a QIAquick spin column in a provided 2 mL collection tube.
3. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30-60 seconds.
4. Discard flow-through. Place the QIAquick column back into the same tube.
5. To wash, add 0.75 mL Buffer PE to the QIAquick column and centrifuge for 30-60 seconds.
6. Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 minute. IMPORTANT: Residual ethanol form Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation
7. Place QIAquick column in a clean 1.5 mL microcentrifuge tube.
8. To dilute DNA, add 40 uL Buffer EB or water to the center of the QIAquick membrane, let stand for one minute, then centrifuge the column for 1 minute. Repeat with 15 mL to elute remaining DNA.