Qiaquick® Gel Extraction Kit from Qiagen

This whole procedure works via spin columns, which is highly comfortable and efficient. Isolation and extraction of gel fragments from agarose gels has come a long way. Specifically, in laboratories that have cloning in their daily routine, this critical step has been time-consuming. A large number of kits for the isolation of DNA fragments from gels are now available on the market and it is up to the scientist to choose which one he or she likes best.

This kit comes in an easy to use format. The digested DNA is separated in horizontal agarose gels and visualized under UV light. The respective DNA fragment is cut from the gel and placed into a microcentrifuge tube. It is important the keep the gel slice small by cutting the DNA fragment as precisely as possible. Also avoid long UV exposure as it will damage the DNA. All needed buffers for the DNA isolation are included in the kit. First, one adds three volumes of Buffer QG to the gel slice which is then solubilized in a waterbath at 50ºC. Sometimes the gel slices do not dissolve completely; this most often occurs with higher percentage agarose gels. Normally, I use 1% TBE gels. In case of incomplete solubilization, one can just extend the incubation time and shake the reaction tubes gently several times during this incubation. Long gel slices also do better when they are cut into smaller pieces. After complete solubilization, one gel volume of isopropanol is added and the solution is immediately applied onto a mini spin column. It is important to note that the isopropanol has to be added after solubilization of the gel slice; if it is added earlier, the solubilization will not work properly. During a one minute centrifugation, the DNA will bind onto the column material. The flow-through is discarded, and 500 ul of Buffer QC is added to wash away all traces of agarose. The column is centrifuged for one minute and the flow-through is discarded. In a third centrifugation step, 750 ul of wash buffer PE (can substitute with ethanol) is added and spun through. Then the column is spun another time to ensure complete removal of the buffer. After this step, the spin column is placed into a clean and sterile microcentrifuge tube. For elution of the DNA, an adequate volume of sterile water or TE buffer is added directly to the column and the column is centrifuged one more time for one minute at full speed in a microcentrifuge. I typically elute in 50 ul sterile water, but when the DNA fragment was only faint visible on the gel, I use less water. Now the extracted DNA is in the flow-through and ready for all downstream reactions. The column is discarded.

I use this kit routinely for about 20 gel extractions per week and ten to twelve can be done easily at a time. The fragment sizes I purify range from 200 bp up to 4000 bp. The DNA is usually concentrated enough for downstream applications such as restriction digests, cloning or labeling and use as a probe in hybridizations. The amount of DNA you elute from the columns is highly reliable and depends, of course, on the amount of DNA you see in the gel before the isolation process.