Can I use plasmid DNA from the Wizard® Plus SV Minipreps System
for automated fluorescent DNA sequencing?
For automated fluorescent DNA sequencing, purified plasmid must be within the proper
concentration range for successful automated cycle sequencing. Ideally the concentration
should be 0.2µg/µl but no less than 0.1µg/µl. The Wizard® Plus SV
Minipreps System(a) routinely yields 3.5-5.0µg of plasmid DNA
when using a pGEM® Vector(b) and DH5alpha™
cells in 1.5ml of LB medium. DNA obtained from either EndA- or EndA+ strains of bacteria
using the Wizard® Plus SV System is suitable for automated
fluorescent DNA sequencing including ABI PRISM® BigDye™ terminator
chemistry.
When performing dilutions of the terminator ready reaction mix, it is essential to
dilute with the appropriate buffer (250mM Tris-HCl [pH 9.0], 10mM MgCl2) and
not water. When using high copy number plasmids, >500 bases of readable sequence can be
obtained at 98% accuracy or greater from terminator ready reaction mixes diluted as much
as 1:6. Complete details on the suggested dilutions are provided in the Wizard®
Plus SV Minipreps System Technical Bulletin (TB225).
When using these suggested methods, ethanol precipitation of purified plasmid DNA prior to
DNA sequencing is not required.
(a)Patent Pending.
(b)U.S. Pat. No. 4,766,072.
Wizard is a trademark of Promega Corporation and is registered with U.S.
Patent and Trademark Office.
ABI PRISM is a registered trademark of and BigDye is a trademark of The
Perkin-Elmer Corporation. DH5alpha is a trademark of Life Technologies, Inc. |
