Wizard SV Gel and PCR Clean-Up System from Promega

Protocol Overview

I amplify constant regions from murine immunoglobulins and variable regions from human B cells. After performing a fusion PCR of the amplicons, appropriate sticky ends are generated with EcoRI and XohI restriction enzymes. Since the buffer used in the PCR is not compatible with the optimal cutting conditions of the restriction endonucleases, the PCR products need to be cleaned and eluted with ddH20. Therefore a quick clean-up system is an ideal solution. After successful clean-up and digestion, the insert needs to be ligated into an expression vector. Again, in order to eliminate the endonucleases and to avoid buffer incompatibilities with the (T4) ligase, the insert needs to be cleaned once again. This illustrates once again the need for a fast and reliable system to purify the precious DNA. The procedure is very simple and involves 3 steps: 1) binding of the DNA to a membrane, 2) washing, and 3) elution. In the recommended manufacturer protocol each step takes between 3 and 7 minutes and the entire protocol can be completed in a total time of about 15 minutes. In my experience, the results of the provided protocol can be improved by adding an additional washing step.