University of Washington Department of Biochemistry

DNA Sequencing Facility




HOW TO OPTIMIZE YOUR SAMPLES SO YOU ACHIEVE THE BEST SEQUENCE FROM US
Keep in mind that EVERYTHING except DNA, primer and sequencing reaction mix act as inhibitors of dye-terminator incorporation.
TEMPLATE QUALITY
  • Avoid the use of cesium chloride, phenol, chloroform etc. in template prep.
  • Use plasmid prep kits specifically made for auto-sequencers.
  • When using kits, remove as much of the wash buffers as you possibly can. Add additional spin steps if you have to.
  • Elute plasmid in half the kit-recommended volume of elution buffer provided …NOT TRIS-EDTA
  • Less volume means more concentrated DNA means residual wash buffers will get diluted out in the sequence reaction.
  • With pcr template fragments, keep elution volumes low and concentration high.

    ABI's Template Quality Recommendations for Capillary Electrokinetics

    We've had good success with Qiagen kits.
PRIMER QUALITY
  • Partially degraded primer, while it will work in pcr reactions, works as an inhibitor in the dye terminator incorporation.
  • Keep a dedicated aliquot for sequencing and know the history of every pipette-tip it has seen.
QUANTITATION
SEQUENCING CYCLES
CLEAN-UP

The 3730 sequencer uses electrokinetic injection to load extension fragments.
Residual salt from ethanol precipitation and residual preservatives from columns can compete with extension fragment uptake .

  • DO NOT ethanol precipitate to remove unincorporated dye.
  • Use G-50 columns specifically made for Dye Terminator clean up like ...pharmasia's 'autoseq', columns from princeston separations, edge biosystems. If packing your own columns … use shells that fit into 1.5 ml microfuge tubes and use DNA grade G-50 hydrated at least 4 hours WITH WATER ( NOTE) …. 1 gram to 15 ml water.
  • If preservatives are present in columns, add a wash with de-ionized water.
  • Pack and spin column at 750g for 2 minutes (all versions of Big Dye Terminator)
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