Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1996 Oct 15;24(20):3879-86.
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, L Chiang, F Dietrich, J Mulligan, R W Davis
Affiliations

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

P J Oefner et al. Nucleic Acids Res. .

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.

PubMed Disclaimer

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Proc Natl Acad Sci U S A. 1959 Nov;45(11):1560-8 - PubMed
    1. Anal Biochem. 1983 Feb 15;129(1):216-23 - PubMed
    1. Biopolymers. 1968;6(5):659-79 - PubMed
    1. Biochemistry. 1977 Oct 18;16(21):4651-4 - PubMed
    1. Nucleic Acids Res. 1977;4(5):1513-37 - PubMed

Publication types

-