From 2007.igem.org

QIAquick PCR Purification Kit Protocol

using a microcentrifuge

This protocol is designed to purify single- or double-stranded DNA fragments from PCR and other enzymatic reactions (see page 8). For cleanup of other enzymatic reactions, follow the protocol as described for PCR samples or use the new MinElute Reaction Cleanup Kit. Fragments ranging from 100 bp to 10 kb are purified from primers, nucleotides, polymerases, and salts using QIAquick spin columns in a microcentrifuge.

Notes:

• Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).

• All centrifuge steps are at ≥10,000 x g (~13,000 rpm) in a conventional tabletop microcentrifuge.

1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene. For example, add 500 μl of Buffer PB to 100 μl PCR sample (not including oil).

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 s.

4. Discard flow-through. Place the QIAquick column back into the same tube. Collection tubes are re-used to reduce plastic waste.

5. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.

6. Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 min at maximum speed.

IMPORTANT: Residual ethanol from 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 elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.

IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 μl from 50 μl elution buffer volume, and 28 μl from 30 μl elution buffer. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.