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Nucleic Acids Res. 1996 Oct 15;24(20):3879-86.

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

Author information

1
Department of Biochemistry, Stanford University, CA, USA. oefner@genome.stanford.edu

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.

PMID:
8918787
PMCID:
PMC146200
DOI:
10.1093/nar/24.20.3879
[Indexed for MEDLINE]
Free PMC Article

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