Wizard® SV Gel and PCR Clean-Up System From Promega

Molecular cloning procedures often require decent amounts of pure DNA fragments, usually generated by PCR or enzyme digestion. The Wizard® SV Gel and PCR Clean-Up System is a multipurpose kit that allows fragments of DNA to be extracted from gel slices or directly from small volume PCR or enzymatic reactions. Three sizes of kit are available (10, 50 and 250 reactions) and each kit contains Membrane Binding Solution, Membrane Wash Solution, Nuclease-Free Water, a set of Wizard® SV minicolumns and collection tubes and a protocol. DNA fragments from 100 bp to 10 kb can be extracted using this kit, either from a PCR reaction or from standard or low melt agarose gels made in either tris acetate or tris borate buffer. The manufacturer states that from 10-40 ng of linear, supercoiled plasmid DNA and single stranded DNA can be purified with the kit, although single stranded DNA may show slightly lower yields than expected. While fragments between 100 bp and 9.5 kb may show 80 to 95% recovery, sizes outside this range are also predicted to show significantly lower yields.

The protocol varies only slightly depending on whether the DNA is extracted from a solution or a gel slice. After being excised from the gel, a slice must be weighed in order to calculate how much Membrane Binding Solution is required to dissolve the slice. This can take time if processing many samples at once, however, it is possible to reduce this by estimating the weight of a standard slice and thereafter, adding the amount of solution appropriate for this standard slice to all your samples. After the addition of the Membrane Binding Solution, the tube is mixed carefully and placed at 50-65ºC to melt the agarose. This takes around 10 minutes, and careful inspection of the tube is required to make sure no solid agarose remains before proceeding – any solid gel will block the column and prevent recovery of the sample. If DNA is to be extracted from a PCR solution, the first step is simply to add an equal volume of Membrane Binding Solution to the tube. In both cases, the next step is to bind the DNA to the silica material in the column. This may be achieved either by a short centrifugation step or by using a vacuum manifold to pull the solution through the column. The bound DNA is then cleaned in the same way by 2 washes with Membrane Wash Solution. The columns are centrifuged briefly to remove any residual Wash Solution and the DNA is eluted by a final spin step with approximately 50 µl Nuclease-Free water.

It should be noted that the vacuum procedure requires the purchase of additional equipment (Vac-Man® Jr. and Vac-Man® manifolds, available from Promega) and still relies on centrifugation for the final elution of DNA. However, this protocol allows up to 20 samples to be bound and washed at once without going back and forth to the centrifuge, which may be very desirable for higher throughput researchers.

I have used the Wizard® SV Gel and PCR Clean-Up System to extract DNA from both gel slices and PCR reactions with satisfactory results. The kit is extremely simple to use, and when processing small numbers of samples (<10) takes no more than 20 minutes. I have tried a range of DNA sizes, and found that I was able to purify decent amounts of DNA from fragments within the range recommended by the manufacturers as well as being able to purify large linearised plasmids (around 13 kb) without a significant drop off in yield. I have used the DNA for transcription and ligation reactions, sequencing and restriction digests without any problems.