DNA and Primer Requirements
- Sequencing and Electrophoresis (Submit template and primer together)
- Purification and Electrophoresis (Submit post-cycle sequencing reactions)
- Capillary Separation (Submit samples for electrophoretic separation only)
Template purity is a key contributor to the quality of the resultant sequence data. It is recommended to use DNA templates that have been post-extraction purified using a column-based DNA purification kit that ensures residual RNA, salt, protein and other contaminating chemicals are removed.
Using the correct amount of template is critical for achieving high quality results. Too little or too much DNA will reduce the length of read and the quality of base calls. Refer to suggested concentrations in the sample submission pdfs for the recommended amount of template. The addition of more than 6 µl of neat template is not recommended as less concentrated templates may produce lower quality sequence reads.
Primer design is very important, as is primer quality. Refer to the primer recommendations within sample submission pdfs. It is recommended to make fresh dilutions of primers at 5 µm every three months.
Post-cycle sequencing purification
Removal of unincorporated dyes from the sequencing reaction prior to electrophoresis is crucial to prevent the presence of dye blobs in the trace, which can obscure correct base call assignment. The Centre uses Sodium Acetate/Ethanol Precipitation purification methods.