PCR PRODUCTS

We can sequence PCR products having a wide size range. It is critical that the product migrate as a single band by gel electrophoresis and be very pure (see below). The quantity of PCR product required for optimal sequencing depends upon the length of the product (see below). Very small PCR products (e.g. < 250 bp) usually require small quantities of DNA (usually 50-100 ng total).

PCR DNA Purity

The DNA should be free of the amplification primers used for PCR, RNA, salt, EDTA, protein, and ethanol. Prior to sequencing, PCR products should be purified by spin column (e.g. Qiagen QiaQuick), preparative agarose gel electrophoresis, or Sephadex chromatography. Shrimp alkaline phosphatase (SAP) combined with exonuclease I also works well for primer removal. The product should run as a single band on an agarose gel. We recommend Qiagen gel purification kit for extracting DNA from gels.

PCR Product Quantity

For PCR products (< 1 kb), we required between 50-100 ng/µl. Please submit at least 10ul per reaction in water or 10mM TE. For larger PCR products (1 - 3 kb), please submit DNA at a concentration of 100 ng/µl. While sequencing can work well with the same primers that were used for PCR amplification, reactions failures may occur and a nested primer is worth considering. The most common cause of PCR product sequencing failure is residual amplification primers in the sample.