Template Preparation

The quality of sequencing results is directlyrelated to the quality of the template. ABI recommends a minialkaline-lysis/PEG precipitation procedure (the Core can supplyinformation on this protocol). ABI also has a plasmid preparation kit whichthey recommend. Several other companies market similar kits. Most of these willwork for automated sequencing. You should experiment and use the method that works best in your lab.

The Core can also provide information on purifying PCR products forsequencing.

NOTE: EDTA is a potent inhibitor of the sequencing reaction. Template in buffer with 1 mM EDTA is acceptable. The final concentration in thesequencing reaction(20 μl) will then be 0.5 mM or less depending on the volumeof template added. A final EDTA of 1 mM or higher will completely inhibitthe reaction.

Possible Template Problems

Template contamination by cellular constituents due to poor technique.

Template degradation - nuclease contamination.

Improperly quantitated template - every template should be quantitated by an OD 260 and visualized on a gel.

Excess salt in the template from purification protocol - 40 mM salt severlyinhibits the sequencing reaction.

EDTA in the template - 1 mM EDTA completely inhibits the sequencing reaction bychelating critical cations.

Multiple templates in the preparation - actually occurs frequently in cloningexperiments where precautions for clonal identity are not taken; results inmultiple peaks per nucleotide position.