The quality of sequencing results is directly related to the quality of the template. ABI recommends a mini alkaline-lysis/PEG precipitation procedure (the Core can supply information on this protocol). ABI also has a plasmid preparation kit which they recommend. Several other companies market similar kits. Most of these will work 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 for sequencing.
NOTE: EDTA is a potent inhibitor of the sequencing reaction. Template in buffer with 1 mM EDTA is acceptable. The final concentration in the sequencing reaction(20 ul) will then be 0.5 mM or less depending on the volume of template added. A final EDTA of 1 mM or higher will completely inhibit the 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 severly inhibits the sequencing reaction.
EDTA in the template - 1 mM EDTA completely inhibits the sequencing reaction by chelating critical cations.
Multiple templates in the preparation - actually occurs frequently in cloning experiments where precautions for clonal identity are not taken; results in multiple peaks per nucleotide position.
These pages are maintained by Joe Forrester (Updated: 09/01/01)
Send comments to: (firstname.lastname@example.org)