Qiagen’s QIAquick Gel Extraction Kit is highly effective for extracting high purity, double-stranded DNA from either PCR reactions or agarose gels following gel electrophoresis. In either case, the procedures are very similar, and by following the manufacturer’s protocols exactly, we find that yields of 50-80% are regularly obtained. The kits are also highly effective at removing impurities – particularly primers and unincorporated nucleotides from PCR reactions.
The kits consist of several buffers and DNA-binding columns; all steps are carried out by adding the appropriate reagent, then spinning the columns at >10,000 x g. Up to 10 ug of DNA can be purified from each column, although the columns are not recommended for purifying fragments less than 100 bp in length (maximum recovery for those is only ~20%). The DNA to be purified is combined with an acidic buffer, containing optimized salt concentrations, and then placed onto the column and the DNA is adsorbed onto the membrane. DNA from PCR reactions can be purified straight away (assuming only a single PCR amplicon is present); DNA in agarose gels is extracted by melting the gels at 55oC for 10 minutes in the acidic buffer. Care must be taken, however, to ensure the buffer remains acidic (the Qiagen buffer contains an internal color indicator - yellow at acidic pH, pink at basic pH). Although in our hands the buffer has never changed pH following gel extraction, if necessary, the pH can be adjusted by adding a weak acid.
Once placed onto the column, the buffer allows adsorption of >95% of the DNA; small fragments such as primers, primer-dimers and unincorporated nucleotides do not bind to the column. These DNA fragments are removed by washing the column; further washes also remove other impurities such as enzymes, ethidium bromide, dyes, glycerol, etc. It is important to remove all traces of wash buffer, as residual buffer components can interfere with subsequent digestion and PCR reactions.
The most critical step of the procedure, we find, is the elution of DNA off the column. Qiagen recommend the use of their patented “EB” buffer (a tris-buffered basic solution), but in practice, we have found that eluting the DNA in sterile water works equally well. Qiagen strongly recommend eluting into small volumes (30-50 ul), and that the eluent should be placed directly onto the center of the column membrane and incubated for at least 1 minute prior to elution by centrifugation. We find that this protocol results in reproducibly high yields of good quality, clean DNA, which we have used for restriction digests, ligation and PCR with no problems. The entire purification protocol generally takes no longer than 20-25 minutes.
In summary, the Qiagen Gel Extraction Kit provides a rapid, low-cost, reproducible and sensitive method to purify DNA to the high level required for many molecular reactions. Although this is a method which lends itself to “short-cuts”, we find that the protocol should be followed meticulously for maximum DNA yields. Yield and purity are verified by running a small volume on an agarose gel, and quantifying by spectrophotometry.