One of the most popular kits in a molecular biology lab, besides DNA miniprep kit, is a PCR purification kit. In our lab, we extensively use Qiagen’s QIAquick PCR Purification Kit which utilizes spin column technology employed in combination with a microcentrifuge. Alternatively, the microcentrifuge can be replaced by a vacuum, but we find it extremely convenient to use a microcentrifuge. The kit is aimed at rapid purification of single- or double-stranded DNA fragments from a PCR reaction. Fragments ranging from 100 bp to 10 kb can be purified from primers, nucleotides, polymerases, and salts. Various buffers are used in order to maximize DNA recovery and eliminate impurities. We usually perform this purification step before restriction-digesting the DNA.
The protocol is very simple. First, three volumes of Buffer QG are added to one volume of the PCR sample and vortexed. Next, one volume (of the initial sample volume) of isopropanol is added and the mix is placed in a QIAquick column and centrifuged for 30–60 s at 13,000 rpm. The flow-through is discarded and the column washed with 0.75 ml Buffer PE and centrifuged for 30–60 s at 13,000 rpm. The flow-through is discarded again followed by an additional 1 minute of centrifugation at 13,000 rpm to completely remove residual ethanol from Buffer PE. The column is then transferred to a clean tube and the DNA eluted with 50 µl Buffer EB. The elution buffer EB should be applied to the center of the column. It is advisable to let the column stand for 1 min before centrifuging 1 minute at 13,000 rpm in order to maximize the yield.
Elution efficiency is dependent on pH, with a maximum efficiency between pH 7.0 and 8.5. If the DNA is eluted in water, it must be within this pH range; all DNA eluted in water should be stored at –20ºC. On rare occasions, DNA fragments eluted with the QIAquick Kit contain denatured single-stranded DNA (ssDNA) which appears as a smaller band on a gel. This is a rare event and eluting DNA in 10 mM Tris buffer containing 10 mM NaCl can allow renaturation of DNA.
While all buffers are ready to use, ethanol (96-100%) needs to be added to the PE buffer before the first use. The kit comes in two sizes: 50 columns or 250 columns. We buy the 250 one as it is much cheaper per column.
Similar products (JetquickPCR from Genomed and Nucleospin PCR from Clontech) that use the silica membrane and spin procedure have similar ease of handling, purity and preparation time (5 – 10 min). However, they differ in the size of the DNA that can be purified: 50 bp – 20 kb for Jetquick PCR, 70 bp – 10 kb for Qiaquick PCR, and 300 bp – 5 kb for Nucleospin PCR. The capacity per prep is 20 micrograms for Jetquick PCR and 10 micrograms for Qiaquick PCR and Nucleospin PCR.