The purity and concentration of the DNA template are the two most critical factors for obtaining good quality sequencing data. The increased sophistication of modern sequencing instruments such as our ABI3730xl allows for improved DNA sequencing but it also increases sensitivity to contaminants in DNA samples. A properly prepared and purified DNA template will result in maximal read lengths, higher sample score values and fewer errors, thus ultimately a quicker turn-around-time.
Plasmid templates can be affected by a variety of contaminants such as cellular components (e.g. RNA, proteins, and polysaccharides), salts, organics or ethanol from commercially-available kits or precipitation method. Below are suggestions for producing a high quality preparation of plasmid DNA for a sequencing reaction. When eluting your samples, we recommend eluting in molecular-grade water.
RNA: The presence of RNA in a sample can interfere with proper quantification of DNA when using a UV spectrophotometer. Similar to DNA, RNA absorbs 260nm light and can result in overestimation of DNA quantity leading to poor sequencing reads.
SALTS: The presence of salts can decrease the processivity of the DNA polymerase used in the sequencing reaction. Care should be taken when precipitating DNA with alcohol to ensure that supernatants are completely removed and sufficient washing with 70% ethanol is performed.
EDTA: EDTA is a chelating agent and can sequester the magnesium required by the polymerase for the sequencing reaction thereby inhibiting its activity. To ensure EDTA is not interfering with the reaction is to provide your samples in sterile nuclease free water.
ETHANOL: The presence of low amounts of ethanol (10%) often causes the sequencing reaction to fail. Take care to avoid transferring ethanol from buffers used in isolation kits to the final elution and to completely drying precipitated DNA samples.
PHENOL: The presence of phenol following DNA extraction will denature the DNA polymerase used for the sequencing reaction. If using a phenol-chloroform extraction protocol take extra care to collect only the aqueous phase.
Sequencing of PCR products can be affected by contaminants such as unused primers and unincorporated dNTPs. Unused PCR primers can result in multiple peaks and a ‘dirty’ sequence read while excessive dNTPs can lead to an unbalanced dNTP/ddNTP ratio, resulting in potentially poor peak height. Below are some suggestions for generating a high quality PCR product for use in a sequencing reaction.