DNA samples commonly run at the Genomic Core Facility are PCR-generated products, plasmids, bacteriophage inserts or cosmids. Purity is the critical part of sequencing-if the templates are not of the utmost purity the sequence results will be disappointing. For plasmid preps, we find that Sigma, Quiagen and Promega kits provide consistant purity, yielding quality sequence data. For PCR products, we have had very good results using the High Pure PCR Product Purification kit from Roche, and also with ExoSAP-IT from USB Corpororation. Important!! Do not use Tris/EDTA (TE) buffer to redissolve DNA or primers! EDTA chelates the Mg++ that is required for the sequencing reactions. Phenol from alkaline lysis mini preps is not tolerated at all in the sequencing reaction. Similarly, incomplete removal of cellular components such as RNA, proteins, polysaccharides, and chromosomal DNA will adversely affect your sequencing results. Residual ethanol in the template can also lead to a failed sequencing reaction. A thoroughly dried column prior to the elution of DNA is imperative.

The table below shows the concentration needed of template needed.  A picture of the agarose gel the template was run on is also required. Please note the volume of template used in the lanes on the gel.