A rapid and simple method for the isolation of high molecular weight cellular and chromosome-specific DNA in solution without the use of organic solvents

Longmire, Jonathan L.; Albright, Kevin L.; Lewis, A.K.; Meincke, Linda; Hildebrand, C.E.

doi:10.1093/nar/15.2.859

Cited by 32 publications

(9 citation statements)

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“…Virus pellets were lysed by the addition of SDS and proteinase K to final concentrations of 0.1% and 100 ~tg/ml, respectively. Tubes were inverted gently and incubated at 37 °C for 3 to 5 h. Samples were dialysed and concentrated, using an autoclaved Schleicher and Schuell Model UH020/2A microdialysis apparatus at room temperature, against four changes (60 min each) of 20% (w/v) polyethylene glycol (PEG) 8000 (Sigma) in sterile Tris-EDTA (TE) buffer (Longmire et al, 1987). Sample volumes decreased approximately 10-fold during PEG dialysis.…”

Section: Methodsmentioning

confidence: 99%

The Herpes Simplex Virus Type 1 Alkaline Nuclease is Not Essential for Viral DNA Synthesis: Isolation and Characterization of a lacZ Insertion Mutant

Weller

Mr²,

Shao³

et al. 1990

Journal of General Virology

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Section: Methodsmentioning

confidence: 99%

The Herpes Simplex Virus Type 1 Alkaline Nuclease is Not Essential for Viral DNA Synthesis: Isolation and Characterization of a lacZ Insertion Mutant

Weller

Mr²,

Shao³

et al. 1990

Journal of General Virology

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“…Crude HSV virions were isolated as described previously (6), except that Vero cells were used instead of RK cells and the sucrose gradient was omitted. HSV DNA was isolated by using a procedure based on the method of Longmire et al (18). Virus pellets were lysed by the addition of sodium dodecyl sulfate and proteinase K to final concentrations of 0.1% and 100 ,ug/ml, respectively.…”

Section: Methodsmentioning

confidence: 99%

An ICP6::lacZ insertional mutagen is used to demonstrate that the UL52 gene of herpes simplex virus type 1 is required for virus growth and DNA synthesis

Goldstein

Weller

1988

J Virol

114

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An insertional mutagen was developed which consists of the lacZ gene of Escherichia coli under the control of the regulatory elements of the large subunit of ribonucleotide reductase (ICP6). This ICP6::lacZ cassette was used to create a mutation in a gene designated UL52 (D. J. McGeoch, M. A. Dalrymple, A. Dolan, D. McNab, L. J. Perry, P. Taylor, and M. D. Challberg, J. Virol. 62:444-453, 1988), which is predicted to encode a 114,000-molecular-weight protein. To isolate and propagate this mutant, we generated a cell line, BL-1, by cotransfection of Vero cells with pSV2neo and a plasmid containing the herpes simplex virus type 1 KOS strain BamHI L fragment (coordinates 0.708 to 0.745). An ICP6::lacZ insertion mutant, hr114, was capable of growing in BL-1 cells but not in normal Vero cells. In addition, hr114 was defective in the synthesis of viral DNA and late proteins; however, this mutant appeared to exhibit normal early gene expression. Thus, the results presented in this report show that the UL52 gene product is required for viral DNA synthesis. Furthermore, our studies indicate that the ICP6::lacZ cassette will provide a useful tool for obtaining mutants of other herpes simplex virus genes.

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“…Several other non-toxic extraction procedures have been published, but require either extensive dialysis (1) or the use of filters (2). A rapid, safe and inexpensive method was developed to simplify the deproteinization procedure.…”

mentioning

confidence: 99%

A simple salting out procedure for extracting DNA from human nucleated cells

1988

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One of the obstacles encountered when extracting DNA from a large number of samples is the cumbersome method of deproteinizing cell digests with the hazardous organic solvents phenol and isochloroform. Several other non-toxic extraction procedures have been published, but require either extensive dialysis (1) or the use of filters (2). A rapid, safe and inexpensive method was developed to simplify the deproteinization procedure. This method involves salting out of the cellular proteins by dehydration and precipitation with a saturated NaCl solution.Buffy coats of nucleated cells obtained from anticoagulated blood (ACD or EDTA) were resuspended in 15 ml polypropylene centrifugation tubes with 3 ml of nuclei lysis buffer (10 mM Tris-HCl, 400 mM NaCl and 2 mM Na2EDTA, pH 8.2). The cell lysates were digested overnight at 37°C with 0.2 ml of 10% SDS and 0.5 ml of a protease K solution (1 mg protease K in 1% SDS and 2 mM Na2EDTA). After digestion was complete, 1 ml of saturated NaCl (approximately 6M) was added to each tube and shaken vigorously for 15 seconds, followed by centrifugation at 2500 rpm for 15 minutes. The precipitated protein pellet was left at the bottom of the tube and the supernatant containing the DNA was transferred to another 15 ml polypropylene tube. Exactly 2 volumes of room temperature absolute ethanol was added and the tubes inverted several times until the DNA precipitated. The precipitated DNA strands were removed with a plastic spatula or pipette and transferred to a 1.5 ml microcentrifuge tube containing 100-200 pl TE buffer (10 mM Tris-HCl, 0.2 mM Na2EDTA, pH 7.5). The DNA was allowed to dissolve 2 hours at 37°C before quantitating.The DNA obtained from this simple technique yielded quantities comparable to those obtained from phenol-chloroform extractions. The 260/280 ratios were consistently 1.8-2.0, demonstrating good deproteinization. Restrictions were performed using a number of different enzymes requiring high, medium or low salt concentrations, all resulting in complete restriction. This procedure has been used in our laboratory on several thousand blood samples for parentage, population and forensic studies. This technique is used with our nonisotopic hybridization procedures (3) rendering the entire process of RFLP analysis free of toxic materials.

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A rapid and simple method for the isolation of high molecular weight cellular and chromosome-specific DNA in solution without the use of organic solvents

Cited by 32 publications

References 0 publications

The Herpes Simplex Virus Type 1 Alkaline Nuclease is Not Essential for Viral DNA Synthesis: Isolation and Characterization of a lacZ Insertion Mutant

The Herpes Simplex Virus Type 1 Alkaline Nuclease is Not Essential for Viral DNA Synthesis: Isolation and Characterization of a lacZ Insertion Mutant

An ICP6::lacZ insertional mutagen is used to demonstrate that the UL52 gene of herpes simplex virus type 1 is required for virus growth and DNA synthesis

A simple salting out procedure for extracting DNA from human nucleated cells

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