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.