Residual salts, organic chemicals or incomplete removal of cellular components such as RNA, proteins, polysaccharides, and chromosomal DNA will all affect the template quality. Applied Biosystems also states that CsCl banded DNA works well if all the CsCl and EtBr have been completely removed via dialysis. In addition, many other commercially available purification kits prepare plasmid DNA suitable for cycle sequencing. Higher concentrations of EDTA or other impurities will inhibit the PCR reaction and result in little or no readable sequence. The DNA concentration of the template is very important and should be accurately determined by Abs. 260nm.
Many laboratories also run aliquots of their templates on agarose gels prior to sequencing.
This is often very helpful in detecting contaminating DNAs or RNAs and getting accurate quantitation. PCR product templates should contain only one band or be gel purified. They must also be completely free of other oligonucleotides prior to DNA sequencing. The DNA Sequencing Facility offers a limited number of tested sequencing primers for sale . As with the DNA template, an accurate DNA concentration of the primer is required.