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A cost-effective plasmid purification protocol suitable for fluorescent automated DNA sequencing

O U Beg1, R G Holt

  • 1Molecular Biology Core Facility, Division of Biomedical Sciences, Meharry Medical College, Nashville, TN 37208, USA. Begobu36@ccvax.mmc.edu

Molecular Biotechnology
|May 21, 1998
PubMed
Summary

A new, cost-effective method reliably produces high-quality double-stranded plasmid DNA. This DNA is suitable for automated sequencing, achieving over 700 accurate bases per run.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Automated DNA sequencing requires high-quality plasmid DNA.
  • Existing plasmid purification protocols may not be optimal for automated methods.
  • Reproducibility and cost-effectiveness are crucial for widespread adoption.

Purpose of the Study:

  • To develop a cost-effective, reliable, and reproducible method for producing double-stranded plasmid DNA.
  • To optimize plasmid DNA purification for automated sequence analysis.
  • To ensure the quality of purified plasmid DNA meets the demands of modern sequencing technologies.

Main Methods:

  • Modification of a previously described plasmid purification protocol.
  • Application of the modified protocol for DNA preparation.

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  • Utilizing a dye-terminator sequencing protocol for analysis.
  • Assessing DNA quality and sequencing accuracy.

    • Main Results:

    • The developed method yields good-quality double-stranded plasmid DNA.
    • DNA prepared using this protocol is suitable for automated fluorescent sequencing.
    • Sequencing runs typically produced over 700 bases with >99% base-calling accuracy.
    • The protocol is cost-effective, reliable, and reproducible.

    Conclusions:

    • The modified plasmid purification protocol is effective for generating high-quality DNA for automated sequencing.
    • This method offers a reliable and reproducible approach for molecular biology laboratories.
    • The protocol enhances the efficiency and accuracy of automated DNA sequencing workflows.