PCR is a routine technique in any molecular biology laboratory. A rapid method for purification of PCR product is, therefore, greatly appreciated in such laboratories. Novagen’s SpinPrep™ PCR Clean-up Kit is made for such a purpose. It helps in removal of salts and primers following PCR, thus generating a pure product. Purification of PCR products using this kit is extremely quick (approximately 10 min.), making it suitable for routine use. The kit is based on a spin-column technique wherein the DNA binds to the membrane of a column. The bound DNA is then washed with an ethanol-containing buffer and later eluted with a low salt buffer or water.
The kit contains a Binding Buffer which is first added to the PCR reaction. If an oil overlay has been used in the PCR, then the solution should be transferred to a separate vial, avoiding oil carry-over. However, small amounts of oil do not interfere in the purification procedure. The DNA-to-Binding Buffer ratio is 1:4. The DNA-Binding Buffer solution is then added to the DNA purification column supplied with the kit. By spinning the column at high speeds, the solution passes over the membrane and the DNA binds to the membrane. The flow-through is generally discarded. However, I have observed that repeating this step two to three times (i.e. re-loading and spinning the flow-through) significantly increases the yield of pure DNA. Moreover, I generally add 2 or 3ul of TrisHCl pH 7.5 to the PCR mixture prior to loading onto the column and this sometimes increases the DNA binding to the column, as suggested in the protocol troubleshooting section.
After the binding step, I pass some more Binding Buffer over the membrane, as recommended. I follow this up by preparing the Wash Buffer supplied with the kit. Wash Buffer is supplied as a concentrate that must be diluted with absolute ethanol prior to first use. After the washing step, I spin the column again to remove residual ethanol and then elute the DNA either with the supplied Elution Buffer or in DNase-free water. The Elution Buffer or water should be pre-warmed (to 600C) before use. I personally recommend water for the elution as the eluted DNA can then be directly used in several downstream applications where salt interference can become a problem.
Using this kit, I have processed up to 6 samples at a time. The length of PCR product in my experiments has ranged from about 200bp to 2.4 kb. The purified product has directly been used for sequencing and restriction digestion for cloning; I have always obtained satisfactory results.
The entire procedure takes nearly 10 min and can take a little more time if the binding step is repeated. The overall recovery using this kit is nearly 80% in my experiments, but can probably vary depending upon the size of the DNA. All the material required for DNA purification is provided with the kit including the collection tubes. Separate collection tubes for the eluted DNA are also provided.
The kit comes in two pack sizes of 20 and 100 reactions. Overall, I am completely satisfied with the performance of the kit in removing unused primers. The eluted DNA is essentially free of salt and unincorporated dNTPs. In fact, I use this kit on a routine basis with every PCR reaction I carry out.
Vikas Jain
Research Scholar
Indian Institute of Science
Molecular Biophysics Unit