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J Biomol Tech. 2007 February; 18(1): 18.
PMCID: PMC2291946

P51-T Optimizing Heat Lysis for High-Throughput Sequencing of Various Plasmid Libraries

Abstract

Heat lysis is a very reliable method for high-throughput labs to decrease costs and increase throughput with little sacrifice in quality of sequence data compared to typically used two-plate or magnetic-bead DNA purification methods. Laboratories employing this procedure on high-copy plasmids, using a sequencing reaction with a 1/8th Big Dye (Applied Biosystems) dilution, have reported generating data with 85% of all wells having read lengths of at least 600 bases with quality value (QV) 20 or above.1

We have adopted this procedure in our laboratory and optimized it for sequencing from various in-house cDNA and genomic shotgun libraries cloned into high-copy plasmids, as well as for libraries constructed outside of our sequencing center. To fully develop the procedure, we utilized a number of culture plates, testing different plate volumes, well shapes, and growth times. Also, we tested various volumes of resuspension buffer in order to generate the highest sequencing success rates and read lengths. To further cut costs, we optimized the sequencing reactions by testing various dilutions of Big Dye and template amounts.

Based upon over 55,000 reads, we have been able to consistently generate sequencing results with average success rates of 90–95% and read lengths of over 700 bases with QV 20 or above. Our past protocol employed a standard two-plate DNA preparation method with a 1/16th Big Dye dilution in the sequencing reaction. In contrast, we now use the optimized heat lysis protocol combined with a 1/32nd Big Dye dilution. These changes have increased throughput and produced the high-quality sequencing results stated above, yet reduced our consumables cost by over 55%.

Notes

Key to Abstract Numbering
Prefixes: P, Poster; RG, Research Group; SP, Scientific Session Presenter; EP, Educational Session Presenter.
Following the hyphen is the designated presentation day: S, Sunday; M, Monday; T, Tuesday.

REFERENCE

1. Ganguly T, Chen P, Teetsel R, Zhang LP, Papaioannou E, Cianciarulo. High-throughput sequencing of high copy number plasmids from bacterial cultures by heat lysis. Biotechniques 2005;39:304–308. [PubMed]

Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities