A freeze-squeeze method for recovering long DNA from agarose gels

Thuring, R.W.J.; Sanders, J.P.M.; Borst, Piet

doi:10.1016/0003-2697(75)90739-3

Cited by 455 publications

(152 citation statements)

References 16 publications

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“…The resulting fragments were separated on 0.6% agarose gels, removed by freeze-squeeze (20), and precipitated with ethanol. DNA fragments (hr 0.74, 38 fmol; hr 0.26, 53 fmol; wt 0.74, 47 fmol; wt 0.26, 60 fmol) were mixed in four combinations as indicated, in 23-jl ligase reaction mixtures containing 0.11 unit of T4 polynucleotide ligase, and incubated at 00 for 2 hours.…”

Section: Methodsmentioning

confidence: 99%

Genetic analysis of host range mutant viruses suggests an uncoating defect in simian virus 40-resistant monkey cells

Wilson¹

1977

Proc. Natl. Acad. Sci. U.S.A.

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Host range mutations that permit simian virus 40 (SV40) to grow with increased efficiency on SV40-resistant monkey cells have been-positioned within the viral B/C gene by a mapping method that relies on the coupling of specific DNA fragments. Pairs of restriction endonucleases that each cleave SV40 DNA at only one site were used to generate pairs of specific DNA fragments. Corresponding pairs of fragments were purified from host range mutant and wild-type DNA and joined in known combinations to determine the location of the host range mutations. The map position of the host range mutations was confirmed by using the same technique to generate and couple geneticall mar ed viral DNA fragments to produce the predicted double mutants. Three different double mutants were constructed that carry both host range and temperature-sensitive A mutations. The mutations in three independently isolated host range mutant viruses are located at very close, perhaps identical, sites, because no wild type viruses were produced from the cell-mediated repair of pairwise heteroduplexes between them. The location of these host range mutations suggests that their phenotype results from mutational alteration of the major capsid protein, the product of the B/C gene.In addition it was demonstrated that monkey cells can efficiently join appropriate pairs of restriction endonuclease fragments intracellularly to produce infectious genomes. That reaction has been partially characterized. The general utility of fragment coupling (in vitro and in vivo) and heteroduplex repair for constructing and analyzing multiple mutants of SV40 is discussed.Virus growth in its host cell involves a number of stages at which obligatory interactions between cellular and viral components occur. A defect in either interacting component can block growth of the virus. Whereas most studies have focused on the viral components by analysis of abortive infections by viral mutants, several recent investigations have sought to define the relevant cellular components in an analogous way by analysis of abortive infections of mutant host cells. This approach has been used extensively in bacteriophage systems (ref. 1 and included references), but is still in its infancy in animal virus systems (2-5). Recently, we reported the isolation of cells that are resistant to simian virus 40 (SV40) from a normally permissive monkey cell population (5). Physiological analysis of SV40 infection of these resistant cells demonstrated that they all are still fully sensitive to infection by SV40 DNA and that they adsorb and eclipse SV40 virus normally. Thus, the cellular defect involves an early stage of SV40 infection after adsorption but before full uncoating.Biochemical analyses of such early stages in animal virus infection are hampered by an inherent limitation arising from large ratios of noninfectious to infectious particles (about 100:1 for SV40). Consequently, any studies that follow the fateThe costs of publication of this article were defrayed in part by the payment of pa...

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Section: Methodsmentioning

confidence: 99%

Genetic analysis of host range mutant viruses suggests an uncoating defect in simian virus 40-resistant monkey cells

Wilson¹

1977

Proc. Natl. Acad. Sci. U.S.A.

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“…Probes for non-radioactive Southern blotting were labeled using the ECL Random Prime Labeling System. After electrophoresis, the DNA fragment to be labeled was purified from the agarose gel by the freeze squeeze procedure (Thuring et al, 1975) or using GENECLEAN II.…”

Section: Southern Blottingmentioning

confidence: 99%

Untitled

1997

Yeast

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“…For separation of large fragments, 2 • 106--20 • 106 daltons, only electrophoresis on agarose gels is suitable. However, the isolation of DNA fragments from the gel often rouses problems [1,2]. Frequently compounds that interfere with either the refragmentation by restriction endonucleases or with the synthesis of DNA or RNA by polymerases, co~elute from the gel.…”

Section: Introductionmentioning

confidence: 99%

“…Certain impurities still co-electrophorese with the DNA, while in addition often losses occur by sticking of the DNA to the dialysis bag [2]. (2) Dissolving the gel in KI [5] or NaC104 [6] followed by phenol extraction or column chromatography of the DNA. Since in these cases the complete gel is dissolved, all the agarose has to be eliminated.…”

Section: Introductionmentioning

confidence: 99%