The QIAquick Gel Extraction Kit can be used after many manipulations (restriction digests, PCR, phosphatasing) to rapidly 'clean-up' DNA for downstream applications (ligations, in vitro transcription, etc.). All Qiagen products for DNA purification generally use the same technology and yield extremely high quality DNA. The Qiagen columns used in this kit contain a silica-gel membrane that binds DNA. Once bound, buffers supplied in the kit can be used to remove contaminants and the purified DNA is then eluted in aqueous solution. The binding, washing and elution steps are dependant on salt concentrations and pH which are optimized in the buffers for each particular application.
Purification of DNA from agarose gels first requires excision of the DNA fragment of interest, for instance generated by a restriction digest or a PCR reaction, as a slab of agarose gel. This is then weighed and an appropriate volume of solubilization buffer is added and the suspension is warmed to 50oC for approximately 10 minutes to solubilize the agarose. The solubilization buffer contains a colorometric pH indicator to verify that pH is optimal for DNA binding to the column. If the pH is too high (the buffer turns purple), it can be adjusted with 3M sodium acetate (something I have never had to do in the course of several years using this product).
Once the DNA is in an appropriate state, either a microfuge or a vacuum manifold is required for column loading and washing steps. The maximum capacity for the silica bed in each column is 10 ug and this amount of DNA must be added in less than 800 ul. Further, the upper limit of DNA size is 10 kb (the lower limit is 70 base pairs), although this size and amount is reasonable for most applications. I have always used a microfuge for the loading and washing and all steps take only one minute to spin. The proceedure is simple and involves the removal of eluent from a collection tube that the column sits in and addition of wash solutions to the top of the column. One issue is the complete removal of the ethanol containing wash buffer, although this is clearly stated in the informative protocol, a common feature of Qiagen products.
The product specifications predict 70-95 % recovery, depending upon the application. Approximate quantitation with gel electrophoresis suggests that these yields are regularly obtained. Perhaps more important is the quality of the DNA. My downstream applications are primarily ligations and these always seem to work, suggesting excellent quality DNA. All told this product can produce purified DNA ready for the next application in around 15 minutes. After a few years of trying techniques such as 'phenol sqeezing' and electroelution in dialysis tubing, such a convenience is extremely welcome.
Peter Haggie, Ph.D.
Post-Doctoral Fellow
University of California, San Francisco