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Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 Oct 15;24(20):3879–3886. doi: 10.1093/nar/24.20.3879

Efficient random subcloning of DNA sheared in a recirculating point-sink flow system.

P J Oefner 1, S P Hunicke-Smith 1, L Chiang 1, F Dietrich 1, J Mulligan 1, R W Davis 1
PMCID: PMC146200  PMID: 8918787

Abstract

Based on a high-performance liquid chromatographic pump, we have built a device that allows recirculation of DNA through a 63-microm orifice with ensuing fractionation to a minimum fragment size of approximately 300 base pairs. Residence time of the DNA fragments in the converging flow created by a sudden contraction was found to be sufficiently long to allow extension of the DNA molecules into a highly extended conformation and, hence, breakage to occur at midpoint. In most instances, 30 passages sufficed to obtain a narrow size distribution, with >90% of the fragments lying within a 2-fold size distribution. The shear rate required to achieve breakage was found to be inversely proportional to the 1.0 power of the molecular weight. Compared with a restriction digest, up to 40% of all fragments could be cloned directly, with only marginal improvements in cloning efficiency having been observed upon prior end repair with Klenow, T4 polymerase or T4 polynucleotide kinase. Sequencing revealed a fairly random distribution of the fragments.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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