PCRcleanup

Following PCR, you often want to get rid of the PCR primers and Taq polymerase before the next step. This is necessary for sequencing PCR products or TA cloning. There is no clean up needed following genotyping reactions. PCR reactions are loaded directly on acrylamide gels.

There are several ways to do the clean up:

1. Run PCR product out on an agarose gel (need to use low melting point Seaplaque GTG), cut out band and use beta agarase to digest away the agarose. This method is preferable if your PCR does not provide a single clean band. One other alternative here is to repeat the PCR by increasing the annealing temperature until you do have a clean band. Then the following methods are easier.

2. Use magnetic beads to bind the PCR product and wash away the primers, followed by elution of the PCR product off of the beads. This is quite fast and produces PCR template that is great for sequencing.

3. Use a spin column (such as Millipore ultrafree). The PCR products (>100 bp) are retained on the column while the primers etc are washed through. After several washes (and slow centrifuge spins) the PCR product can be pipetted back off the column membrane.