Plasmid Template Preparation

The quality of the template DNA can have a major impact on the quality of the sequence data you receive. Applied Biosystems states that inadequate template preparation is the most common cause of sequencing problems. For all template preparation methods, be sure to follow the recommended procedures.

Commercial template preparation kits recommended by Applied Biosystems are:

  • ABI Prism mini Prep Kit
  • Qiagen columns
  • AGTC columns
  • Nucleobond AX
  • 5' -> 3' Perfect Prep

Kits that are not recommended are:

  • Bio101 GeneKleen
  • BioRad Prep A Gene
  • Promega Wizard (lot to lot variability is of concern)

Template Concentration:

Plasmids must be supplied to us in water with a concentration of 0.1 to 0.2 ug/ul. TE buffer greatly interferes with the Taq polymerase during cycle sequencing. The concentration must be determined by taking an OD reading.

GC Rich Templates:

GC-rich templates with a GC content greater than 65 percent may be difficult to sequence. If your template is GC rich, please inform us. We can add glycerol and DMSO to your reaction to achieve better sequencing results.

Templates with Homopolymer Regions

When more than 10 of the same base occur consecutively, problems may be observed in the data. With G and C, problems may be observed with fewer than 10 bases, whereas, with A and T, the enzymes can generally read through longer sequences. What happens is that the Taq polymerase enzyme "slips" through this region and your data after the homopolymer is usually obscured.

Other Tips:

Please take note that templates that are not sequencing manually will probably not sequence by automated techniques either.

Please feel free to contact us: Lab: (410) 955-2739 | Fax: (410) 614-7566 | e-mail: syn_seq@jhmi.edu

Copyright © 2005 Johns Hopkins University School of Medicine. All Rights Reserved.

Site design by Academic Web Pages