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TurboPrep II: an inexpensive, high-throughput plasmid template preparation protocol
1Center for Medical Genetics, Marshfield Medical Research Foundation, WI 54449, USA.
Biotechniques
|March 12, 1998
Summary
A new boiling minipreparation protocol significantly reduces costs for large-scale genomic sequencing. This method efficiently prepares high-quality plasmid DNA templates, yielding over 300 bp of readable sequence.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Plasmid DNA template preparation is a costly bottleneck in large-scale genomic sequencing.
- Existing methods often require expensive reagents and significant hands-on time.
Purpose of the Study:
- To develop a cost-effective and efficient protocol for preparing plasmid DNA templates for genomic sequencing.
- To enable high-throughput template preparation by a single individual.
Main Methods:
- A boiling minipreparation protocol was developed for plasmid DNA extraction.
- The protocol was evaluated in both manual and automated configurations.
- Template quality was assessed using radioactive cycle sequencing and polyacrylamide gel electrophoresis.
Main Results:
- The manual protocol allows preparation of 768 templates in 8 hours, with a maximum throughput of 1920 templates.
- Automation increases throughput and yields approximately 60% more DNA.
- The cost per template is less than twenty cents.
- Sequencing yielded over 300 bp of readable sequence from prepared templates.
Conclusions:
- The boiling minipreparation protocol offers a significant cost reduction for large-scale genomic sequencing.
- The method is scalable and adaptable for automation, further enhancing throughput and yield.
- Prepared templates are suitable for various downstream applications including sequencing, hybridization, and host cell transformation.