The Cyclo-Pure Gel Extraction Kit is a spin column based kit designed to isolate DNA from standard agarose gels quickly and efficiently. The procedure allows for the isolation of specific DNA bands for downstream procedures. The kit supplies all of the necessary reagents (except for the elution buffer) and comes with a simple, one page manual. The procedure is not difficult and I have found this kit to be the most reliable for DNA isolation. It cleans 40 bp – 100 kb DNA from TBE or TAE buffered agarose gels.
The protocol is simple and straightforward. The desired DNA band is excised from an agarose gel that has been run in TAE or TBE buffer. The manual does not state a gel percentage requirement but I have extracted DNA from 2% and 1% gels with equal efficiency. Following excision, the gel slice is weighed in order to determine the required amount of Binding Buffer. Once the gel slice is determined, the volume of Binding Buffer is determined by using 3 ml buffer to each gram of gel. For example, if the gel slice weighs 0.1 gram, 0.3 ml of binding buffer will be added. The gel slice and Binding Buffer are added to a microcentrifuge tube and heated at 55ºC for 5 minutes or until the gel slice is completely melted. I have found that it often takes longer than 5 minutes; inverting the tube throughout the 55ºC incubation decreases melting time. My slices tend to be 0.1 – 0.2 g. The solution is then transferred to a spin column and spun at top speed in a microcentrifuge for 30 seconds. The filtrate is discarded and the column is washed with the Washing Solution. The filter is then dried with another high speed spin, for 2 minutes. The DNA can then be eluted in water or TE buffer heated to 60 – 70ºC.
This protocol is very similar to other kits I have tried. However, the one difference I have seen with this kit is that the melting of the agarose is done in a separate tube from the spin column. I have had difficulty when melting in a spin column and have frequently had agarose carryover in my samples. I have also had very low yields when using other kits, especially with smaller DNA fragments. So far, I have not seen this happen with the Cyclo-Pure Gel Extraction Kit. Using this kit, I have purified bands containing DNA concentrations ranging from 40 ng to 240 ng averaging about a 20% loss after purification. Furthermore, I have purified DNA bands ranging from 150 bp to >1 kb. The post-purification loss does increase significantly with the smaller DNA fragments (e.g. 150 bp). However, this is seen in all kits and I had the best recoveries with this kit. I have also used 0.9% and 2% gel pieces with the 2% requiring extended melting times.
Overall, even though there are numerous gel extraction kits available, I think Amresco’s Cyclo-Pure Gel Extraction Kit does the best job with the highest recovery of DNA.
Katrina Stumpf
MS Student and Senior Lab Specialist
McGuire VA Medical Center
Division of Infectious Disease