The QIAEX II Gel Purification Kit can be used for extraction and purification of DNA from either TAE or TBE agarose or polyacrylamide gels. The extraction process is designed such that phenol or ethanol precipitates are not required, instead making use of silica particles to enhance recovery of very small or large DNA fragments. The kit is optimized to recover DNA molecules ranging from as small as 40 bp up to 50 kb and all impurities such as agarose, proteins, salts and ethidium bromide are removed, during a series of washing steps. The kit states a recovery of 60-95% of DNA fragments in a minimum elution volume of 20 ul. The resultant DNA may be used in downstream applications such as restriction digests, labelling, ligation, PCR, sequencing and microinjection. The kit protocol contains a table stating the separation ranges in TAE gels containing different concentrations of agarose, allowing the user to select the optimum percentage gel for purification of their DNA fragment target. I have routinely purified PCR products ranging from 350 bp to 1 kb, all from a 2.0% agarose TBE gel.
The protocol for agarose gel extraction involves first running the DNA to be purified on a suitable gel and then excising the DNA bands into a microcentrifuge tube with a clean scalpel. Each band must then be weighed and suitable volumes of Buffer QXI added (3 – 6 volumes dependant on the size of the DNA fragment and % agarose gel). 10-30 ul of QIAEX II silica particles are then added to each sample and this must be incubated for 10 minutes at 50°C. The high concentration of chaotropic salts in the QXI buffer disrupts hydrogen bonding between sugars of the agarose polymer, thus allowing solubilization of the gel and dissociating DNA binding proteins from the DNA fragments. The buffer is optimized to dissolve both TBE and TAE gels and the 50°C incubation allows for the DNA to absorb to the QIAEX II particles.
The adsorption of DNA to the QIAEX II particles is pH dependant and the efficiency is approximately 95% if the pH is 7.5. The QXI buffer, for this reason, contains a pH indicator and will turn from yellow to an orange or purple color if the pH of the sample exceeds the buffering capacity of QXI. This can easily be corrected by addition of a small volume of 3M sodium acetate, pH 5.0. The adsorption of DNA to the QIAEX II particles removes non-nucleic acid impurities, which remain in the supernatant. After the 10 minute incubation, the sample is centrifuged at maximum speed for 30 seconds and the supernatant removed with a pipette. The sample is then washed with 500 ul of QXI buffer, which serves to remove residual agarose. The sample is then centrifuged again and washed with 500 ul of the ethanol-containing Buffer PE, which efficiently removes all salt contaminants. This wash is repeated and the residual QIAEX II particle pellet is air dried at room temperature for 10-15 minutes in order to remove all residual ethanol. The DNA is eluted by the addition of 20 ul Tris-CL pH 8.5, or H2O, re-suspension of the particles and incubation for 5-10 minutes. Finally the sample is centrifuged for 30 seconds and the supernatant containing the DNA is removed.
I use the kit to routinely purify PCR products for downstream sequencing. The kit has always produced high quality DNA with good yield. The DNA has been used for sequencing reactions and there have been no problems with any impurities or inhibitors that may have been introduced by the kit. The kit is quite expensive if purifying 2-10 ug of DNA, as 30 ul of the QIAEX II particles are required and as a result, the kit will only purify 50 samples and not the 150 stated. However, the individual components can be bought separately allowing those that run out to be individually replaced. I use the kit to purify PCR product from 2.0% TBE gels and the QXI buffer is successful at DNA purification from this source. The pH indicator contained in the buffer is very useful, as the DNA will not adsorb to the silica beads if the pH reaches too high a level, however I have never encountered this problem.
PhD Student
Academic Hematology
Newcastle University