DNA & primer requirements
Instructions for sample submission requirements for services:
Sequencing and electrophoresis (pdf)
Purification and electrophoresis (pdf)
Electrophoretic separation (pdf)
Up to 1000 bases can be sequenced per run however the accurate read length is largely dependent upon a several important factors:
Template purity is a key contributor to the quality of the resultant sequence data. It is preferred that all DNA templates are post-extraction purified using a column-based DNA purification kit that ensures that residual RNA, salt, protein and other contaminating chemicals are removed. ExoSAP-IT for PCR purification and TempliPHI for plasmid amplification routinely produce very high quality DNA for sequencing as does the Alkaline Lysis PEG precipitation method.
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 the tables in the sample submission pdf’s for the recommended amount of template required. The volume of template used should not exceed 6ul as we have found that less concentrated templates produce lower quality sequence reads.
Primer design is very important, as is primer quality. It is recommended to make fresh dilutions of primers at 5um 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 that can obscure correct base call assignment. The Centre uses Sodium Acetate/Ethanol Precipitation or BigDye XTerminator purification methods.