The Promega Wizard SV Gel and PCR Clean-Up System is designed to extract DNA fragments from agarose gels or clean up PCR products. The system has the flexibility to extract from both regular and low melting point agarose prepared in either TAE or TBE buffers. The SV system is a membrane-based system, where DNA is bound to a silica membrane in the presence of chaotropic salts.
We have used this system primarily to purify fragments from agarose gels for sequencing and/or cloning. Once the gel has been run, the band of interest is visualized with a transilluminator and excised from the gel using a sterile scalpel or blade. The agarose slice is transferred to a pre-weighed micro-centrifuge tube, re-weighed. The calculated weight of the agarose slice determines the quantity of the membrane binding solution (4.5M guanidine isothiocyanate; 0.5M potassium acetate) to add to the tube. The agarose and membrane binding solution are vortexed and heated to 50-65oC for approximately 10 minutes or until the agarose is dissolved. At this point, the melted agarose solution is decanted into the membrane-containing filter unit. We proceed at this point with microcentrifuge purification, but vacuum purification can also be performed. The filter unit is placed in a collection tube and the mixture is incubated in the filter unit for 1 minute prior to spinning at 10,000 x g. The flow through is removed and then the filter is washed twice with the membrane wash solution (10 mM potassium acetate, 80% ethanol, 16.7 uM EDTA) via centrifugation. After the washes, the filter unit is placed in a clean microcentrifuge tube. 50l of water is added to the membrane and incubated and incubated for 1 minute. This is then centrifuged again and the DNA is within the eluate. The DNA at this point is ready for subsequent applications or storage at 40C or -200C.
Clearly the beauty of the Promega Wizard SV Gel and PCR Clean-Up System is the rapid and simple nature of the procedure; however, this would be of little benefit if the recovery from agarose was poor. After trying similar systems from Qiagen and Invitrogen with disappointing results, Promega’s system has become our favorite. Using this system to purify DNA, mostly in the 1kbp to 5 kbp range, we consistently obtain a minimum of 75% recovery (as estimated by a gel analysis of the eluted DNA). Another useful feature of this system is the water elution, facilitating downstream applications with complimentary buffers.
Boyd Scott
Senior Research Scientist
Discovery Biology
Vitae Pharmaceuticals