Molecular Biology

DNA Sequencing | Customized Services | Quantitative PCR

CFG DNA Sequencing Services

Template Concentrations | Primer Concentrations | Policy on Repeat Reactions
Quality Control | Standard Primers | Template Preparation

Recommendations for Template Preparation

For successful cycle sequencing, it is crucial that templates be of high purity and appropriate concentration. Possible deleterious contaminants include bacterial proteins and carbohydrates and organic solvents such as methanol, isopropanol, etc. Templates submitted should be accurately quantitated by determining both A₂₆₀ and the A₂₆₀/A₂₈₀ ratio (ratio must be at least 1.8). Submitted templates should be resuspended in water or 10 mM Tris/HCl, not TE, as EDTA will inhibit the activity of the Taq enzyme.

The CFG recommends isolation of plasmid templates by use of one of the commercially available kits (i.e., Qiagen, Promega, etc.). PCR products must be purified to remove residual primers. For PCR products CFG recommends isolation with commercial kits (i.e., Qiagen, Promega, etc.). Because these systems use salts and ethanol, extensive washing of pellets to remove salts and complete drying to remove ethanol is required.

DNA Sequencing | Customized Services | Quantitative PCR