Sequencing

Fifth Step:

The fifth step in the molecular process is sequencing, following these techniques:

• The samples from the freeze and squeeze step are mixed with sequencing reaction mix and buffer, and the same primers that were used during the amplification step in strip tubes
• Once prepared, these strip tubed are placed into the thermocycler and run
• Here, we send these products off to be sequenced at another lab, which will send us the results in the form of digital chromatogram files
• These chromatograms can then be subjected to review and verification – the sequence can be BLASTed to check if it is likely to be the correct product
• Depending on the quality of the sequence (i.e. if there is clear single read, rather than spurious peaks or even double peaks), the sequence may be ready to be used in phylogenetic analyses