Template preparation for Sanger Sequencing

Template Prep and Quality

The starting template DNA is the single most important determinant of the quality of the final sequencing data.

The vast majority of our customers have obtained excellent results using the Qiagen* plasmid DNA kits (QiaPrep, Qiagen-tip 20, Qiagen-tip 100, Qiaprep spin, etc.) or the Qiawell plasmid kits. The QiaPrep and Qiawells are the most reliable way of isolating high quality plasmid DNA suitable for automated fluorescent sequencing, and we strongly recommend their use for template DNA purification.

In addition to the Qiagen procedure, there are two other recommended methods for consistently preparing high quality template DNA:

• Ultracentrifugation in CsCl density gradients often yields excellent template DNA. Following centrifugation, one must carefully remove residual CsCl from the DNA either by dialysis and/or ethanol precipitation (Cs inhibits Taq polymerase).
• The Wizard (formerly Magic) DNA purification system gives good DNA if extra steps are added to the standard procedure. Adding an extra ethanol precipitation at the end greatly improves the reliability of this method for preparing high quality template DNA. Overall the Wizard method appears to be somewhat less consistent than the Qiagen procedure, but in the hands of an experienced user, roughly 90% of Wizard templates generate good sequence data.

Common Mistakes

Although the QiaPrep and Qiawell plasmid kits are very reliable, there are some common mistakes which compromise the final quality of sequence data:

• The isopropanol precipitated DNA is not washed with 70% ethanol to remove excess salt. Wash the DNA pellet at least once as directed with 70% ethanol. Residual salt in the final template will interfere with the activity of Taq polymerase resulting in sequence data which extends less than 200 bases from the primer and exhibits a low signal to noise ratio.
• The template DNA is not dried completely before final resuspension in H₂O or TE. To remove residual ethanol, dry the DNA for 5 min. in a properly operating speedvac. If air-drying is preferred, make sure that the DNA is dry (no fluid in the tube, the DNA pellet doesn't look wet). When air drying, a brief 15 min incubation of the open tube at 65°C is often sufficient to completely dry the DNA. Residual ethanol is very detrimental to Taq cycle sequencing resulting in data with a drastically reduced signal.
• The directions for cell growth are not followed resulting in overloading the Qiagen resin. Usually, a poor yield of plasmid DNA results, presumably due to competition with RNA fragments for binding to the Qiagen resin. Use the recommended quantity of LB broth (don't use Terrific Broth) for cell growth.