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How to purify PCR products?
The PCR product must first be run on an Agarose gel. The target band is excised and then purified using a gel extraction kit. The eluted product should be dissolved in ddH₂O.
Why is Agarose gel purification mandatory for direct sequencing of PCR products?
If gel purification is not performed and only a purification kit is used, it often leads to double peaks or messy peaks in the sequencing results. This is mainly caused by non-specific amplification products or incomplete removal of the original PCR primers. Most so-called PCR "purification kits" primarily recover the product but do not effectively purify it. They certainly cannot remove non-specific amplification products, and often they do not completely remove all PCR primers. These residual primers can participate in the sequencing reaction, causing messy peaks.