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

Primer Concentrations

Please follow the guidelines below when providing your own primers for your sequencing request. We use 1 µL of primer per reaction. Please provide enough primer so that reactions can be repeated, if necessary. Submission of primers that are not at the requested concentration may result in a delay in the processing of your samples.

Primer Design

Based on Applied Biosystems'BigDye® Terminator v3.1 Cycle Sequencing Kit Protocol. Part number4337035 Rev. A
The following recommendations are provided to help optimize primer selection:

• Primers should be at least 18 bases long to ensure good hybridization.
• Avoid runs of an identical nucleotide, especially guanine, where runs of four or more Gs should be avoided.
• Keep the G-C content in the range 30­80%.
• For cycle sequencing, primers with melting temperatures (T_m) above 45 °C produce better results than primers with lower T_m.
• For primers with a G-C content less than 50%, it may be necessary to extend the primer sequence beyond 18 bases to keep the T_m>45 °C.
• Use of primers longer than 18 bases also minimizes the chance of having a secondary hybridization site on the target DNA.
• Avoid primers that have secondary structure or that can hybridize to form dimers.
• Several computer programs for primer selection are available. They can be useful in identifying potential secondary structure problems and determining if a secondary hybridization site exists on the target DNA.

DNA Sequencing | Customized Services | Quantitative PCR