The level of contaminating products in the amplified product has a direct effect on the quality of the final sequencing results. These can be minimized by optimizing the PCR and by using appropriate methods to reduce carryover. To minimize carryover from a previous PCR amplification, use standard recommended procedures for laboratory cleanliness, and either a separate set of pipettes or aerosol resistant tips. Careful selection of PCR primers can reduce primer-dimer artifacts and enhance the selection of the specific template target during sequencing. The quantity of the PCR product is also an important consideration in selecting a sequencing chemistry.
Cycle sequencing protocols require nanogram amounts of template.
See associated product information for specific template DNA requirements. In addition to requiring very pure DNA, Dye Primer Sequenase chemistry requires single-stranded template. There are several ways to accomplish this, including asymmetric PCR and the use of specific enzymes to make the DNA single-stranded. One of the easiest ways is to use a magnetic bead-based solid phase approach, which performs purification and permits the bound DNA strand to be separated from the unbound strand. Note, however, that the use of solid phase purification is limited to PCR products shorter than one kilobase.