Automation-Robotics Product Profiles

Wizard^® SV 96 and
Wizard^® SV 9600
Plasmid DNA Purification System

The Wizard^® SV 96 Plasmid DNA Purification System^(a) allows the purification of up to 96 plasmid minipreps in less than 60 minutes. The procedure can be performed manually or can be adapted for use with automated instruments. The purified plasmid DNA is suitable for use in automated fluorescent sequencing using a number of different chemistries including BigDye™ terminator chemistry.

The SV 96 system contains all the reagents necessary for purification of plasmid DNA, including:

• 96 Well Culture Plates-- allowing growth of up to 96 x 1.5ml cultures at a time
• Lysate Clearing Plates-- providing a rapid alternative to centrifugation for removal of cellular material from bacterial lysates without centrifugation
• DNA Binding Plates-- purifying high quality plasmid DNA  

The Wizard^® SV 9600 System contains Clearing and Binding Plates as well as reagents to purify 9600 (100 x 96) minipreps on a robotic workstation in the most high-throughput setting.

Figure 1. Protocol Overview. Flow diagram of plasmid DNA isolation and purification using the Wizard^® SV 96 Plasmid DNA Purification System and Vac-Man^® 96 Vacuum Manifold. 

Rapid plasmid DNA purification

Plasmid DNA is purified from bacterial cultures using a series of simple steps:

• alkaline-SDS lysis to generate a cleared bacterial cell lysate
• lysate clearing through a Lysate Clearing Plate
• plasmid capture and purification using SV membrane technology

The purified plasmid DNA can be used directly for automated fluorescent sequencing as well as other standard molecular biology techniques.

The system is designed for use with a vacuum manifold such as the Vac-Man^® 96 Vacuum Manifold. When used with the Vac-Man^® 96 Manifold, concurrent lysate clearing and plasmid DNA binding is achieved. The use of a waste trap (vacuum trap not supplied) with the Vac-Man^® 96 Manifold eliminates the need for disassembly during the process to discard the wash solutions.

Versatile system allows manual and automated processing

The Wizard^® SV 96 System is designed to be used either in a manual format or can be adapted for use with automated purification instruments. Protocols are available for use on a number of automated systems including the Beckman Coulter Biomek^® 2000 workstation.

Manual format configuration. Wizard® SV 96 System is designed to allow lysate clearing and DNA binding simultaneously when used with the Vac-Man® 96 Vacuum Manifold. Waste solutions are collected in an external trap (not provided).

Automated processing. The Wizard® SV 96 System is compatible with a number of automated instruments including the Biomek® 2000 workstation.

Unique plate design prevents spraying during sample processing

Elution of DNA samples into small volume 96 well microtiter plates is the preferred method of collection and storage of DNA samples. This can be a problem when using certain 96 well plasmid purification systems. These systems have narrow aperture elution ports on the DNA capture plate, which tend to spray when a vacuum is applied. As a result, centrifugation is required to collect the sample in the well bottom. A unique plate design has been used with the Wizard^® SV 96 System to minimize spraying. The larger aperture and increased length of the elution ports allows eluted DNA to be collected in standard 96 well microtiter plates.

Wizard® SV 96 plate tips commercially available 96 well plate

Figure 2. Plate tip design comparison. Photographs show increased tip aperture and absence of skirt for the Wizard® SV 96 plate tips (top) versus the tips of a second commercially available 96 well plate (bottom).

Highly pure DNA suitable for a variety of techniques

Fluorescent sequencing. The Wizard^® SV 96 System has been designed to purify plasmid DNA suitable for fluorescent sequencing using a number of different chemistries including BigDye™ terminator chemistry. Rigorous quality control procedures ensure that plasmid DNA purified using the Wizard^® SV 96 System meets or exceeds 600 bases with over 98% accuracy using Dye Primer, BigDye™ Primer and BigDye™ Terminator chemistries.

Figure 3. Accuracy by read length for plasmid DNA isolated using the Wizard^® SV 96 System followed by cycle sequencing using Dye Primer, BigDye™ Primer and BigDye™ Terminator kits. pGEM^® 3Zf(+) DNA direct from the Wizard^® SV 96 Elution Plate was used with each of the three sequencing reactions using reaction conditions and cycling parameters as specified by the manufacturers (1-3).

Figure 4. Electropherogram of plasmid DNA sequence following isolation by the Wizard^® SV 96 System and cycle sequencing using BigDye™ Terminator reactions. Results demonstrate greater than 700 consecutive bases analyzed with greater than 98% accuracy of base identity.

Restriction enzyme digests. The Wizard^® SV 96 System provides reproducible yields of high-quality plasmid DNA for use in standard molecular biology applications. Plasmid DNA purified using the Wizard^® SV 96 System has been extensively tested and is guaranteed for use in restriction digestion analysis.

Figure 5. Analysis of restriction enzyme digestion of plasmid DNA. Restriction digestions were performed on three different plasmid DNA preparations (A-C). All digestion reactions included 10 units of enzyme per microgram of plasmid DNA (pGEM^®-3Zf(+) Vector). The reactions were incubated for one hour at 37°C. Lane M. Lambda DNA/Hind III Markers (Cat.# G1711). Uncut control (C, lanes 1, 5 and 9); EcoR I (E, lanes 2, 6 and 10); Sca I (S, lanes 3, 7 and 11); Sca I/Sph I (S/S, lanes 4, 8 and 12).

Automation Platform Protocols

The Wizard^® SV 96 System has been developed for manual use or on selected automated platforms.

Wizard^® SV 96 System Protocol Abstract
Wizard^® SV 9600 System Protocol Abstract
Biomek^® 2000 Laboratory Automation Workstation

1. ABI PRISM^® BigDye™ Primer Cycle Sequencing Ready Reaction Kit with AmpliTaq^® DNA Polymerase, FS Protocol. P/N 403057 Rev. B. (1997).
2. ABI PRISM^® BigDye™ Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq^® DNA Polymerase, FS Protocol. P/N 4303237 Rev. B. (1998).
3. ABI PRISM^® Dye Primer Cycle Sequencing Ready Reaction Kit with AmpliTaq^® DNA Polymerase, FS Protocol. P/N 402113 Rev. B. August, 1995.

pGEM, Vac-Man and Wizard are trademarks of Promega Corporation and are registered with the U.S. Patent and Trademark Office.
BigDye is a trademark of The Perkin-Elmer Corporation.

Products may be covered by pending or issued patents. Please visit our Patent Information for more information.