An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels

Tautz, Diethard; Renz, Manfred

doi:10.1016/0003-2697(83)90419-0

Cited by 526 publications

(224 citation statements)

References 9 publications

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“…Partial digestion yielded, on a 1.5% TAEagaro~e gel, 3 distinct bands ofabout 500, 300 and 200 bp. The 500.bp band, containing non-digested DNA (505 bp) and the DNA digested at the 5' Ncol site (494 bp), was isolated from the gel by the freezesweep method [32], This DNA was digested with BamH! and ligated in pET3d, which was previously digested with BamHl and Ncol and aho purified from the gel, The resulting plasmid was transformed to E, coli strain PC2495, In order to check the nuclcotide sequence of the imert, double-strand sequencing was performed according to instructions supplied with the T7 sequencing kit on plasmid DNA that was isolated according to the boiling method [31] and extensively purified b~, using Prep-a-Gene purification matrix,…”

Section: Methodsmentioning

confidence: 99%

The precursor form of the rat liver non‐specific lipid‐transfer protein, expressed in Escherichia coli, has lipid transfer activity

1992

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The cDNA encoding the precursor form of non-specific lipid-transfer protein (pre-nsL-TP) from rat liver was cloned into the expression vector pET3d. The resulting plasmid was transformed to the Esrherichia colt strain BL21(DE3). After induction of the bacteria with isopropyl./5'-Dthiogalactopyranoside (IPTG) pre-nsL-TP was purified from the bacterial lysat¢ by anion exchange chromatosraphy followed by 8elliltration. From 1 I of culture, 6-7 nag of pre-nsL-TP was obtained, equal to approximately 7% of the cytoplasmic protein. By use of a fluorescence lipid transfer assay, prc.nsL-TP was found to have lipid transfer activity identical to mature nsL-TP.Non-specific lipid-transfer protein; Sterol carrier prolein-2l Lipid transfer; Bacterial expression; Rat liver

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Section: Methodsmentioning

confidence: 99%

The precursor form of the rat liver non‐specific lipid‐transfer protein, expressed in Escherichia coli, has lipid transfer activity

1992

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“…KTT42 chromosomal DNA was digested to completion with BamHI; the single BamHI site in TnphoA is located downstream of the kanamycin resistance marker and the end of phoA. Fragments (6-8 kbp) were size fractionated by gel electrophoresis, purified by the freeze-squeeze technique (Tautz and Renz, 1983) and ligated into the unique BamHI site of pUC19. The construct was confirmed by restriction enzyme digestion and Southern hybridization (not shown) and named pKTT42.…”

Section: Strain Constructionsmentioning

confidence: 99%

Phase variation in tcpH modulates expression of the ToxR regulon in Vibrio cholerae

Carroll

Tashima

Rogers

et al. 1997

Molecular Microbiology

124

126

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SummaryWe evaluated a spontaneous mutant of Vibrio cholerae, which was avirulent in an infant mouse and had reduced expression of cholera toxin and TcpA in response to environmental signals. The toxR, toxS and toxT genes in the mutant were normal, but transcription of toxT was absent. A plasmid expressing wild-type tcpP and tcpH complemented the mutant. The mutation resulted from a frameshift in a string of nine G residues within tcpH; similar slipped-strand mutations in tcpH arose at a frequency of 10 ¹4 during overnight growth and in the majority of colonies by the end of 5 days of growth in ToxR-inducing conditions. Transcription of tcpPH was regulated by temperature and pH independently of ToxR or ToxT. These results suggest that TcpH couples environmental signals (temperature and pH) to expression of the ToxR regulon, and provide a model for phase variation in the co-ordinate expression of cholera virulence factors.

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“…The secondary PCR reactions used the primer combinations o200/odT20N2 or o16/odT20N2 and identical cycle conditions. Resulting PCR products were analyzed in 1.3% agarose gels and the DNA fragments obtained were purified using the method of Tautz and Rentz (1983). Cloning of purified DNA fragments into pGEM-T (Promega) or the Bluescript SKIT vector (Stratagene), M 13 phage DNA isolation, and sequencing were performed using established methods (Sambrook et al, 1989;Ausubel et al, 1991).…”

Section: '-Race and Dna Sequence Analysismentioning

confidence: 99%

TPO1, a Member of a Novel Protein Family, Is Developmentally Regulated in Cultured Oligodendrocytes

Krueger

Gonye

Madison

et al. 1997

Journal of Neurochemistry

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Although the myelin membrane contains only a small set of major proteins, more sensitive assays indicate the presence of a plethora of uncharacterized proteins. We have used an antibody perturbation approach to reversibly block the differentiation of prooligodendroblasts into myelinating cells, and, in combination with a differential screening procedure, identified novel mRNAs that are activated during this period. One cDNA, TPO1, recognizes a 5.5-kb mRNA that is strongly up-regulated in oligodendrocytes after release of the differentiation block and that is expressed at high levels in brain tissue during active myelination. This cDNA represents at least two mRNAs differing from each other in their 5'-termini. The TPO1 cDNA contains an open reading frame of 1,380 bp, encoding a protein of 51 .8 kDa with a predicted pi of 9.1 that contains two regions homologous to nonclassic zinc finger motifs. Subceilular localization studies suggest the enriched presence of TPO1 in spherical structures along the major cytoplasmic processes of oligodendrocytes. TPO1, along with homologues expressed in testis, placenta, and PC12 cells, form a novel family of proteins with multiple hydrophobic domains possibly serving as membrane spanning regions. We postulate that in oligodendrocytes, TPO1 encodes a protein factor involved in myeiin biogenesis. Key Words: TPO1 -Oligodendrocyte development-Myelin-Zinc finger-Membrane protein-OB-fold.

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An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels

Cited by 526 publications

References 9 publications

The precursor form of the rat liver non‐specific lipid‐transfer protein, expressed in Escherichia coli, has lipid transfer activity

The precursor form of the rat liver non‐specific lipid‐transfer protein, expressed in Escherichia coli, has lipid transfer activity

Phase variation in tcpH modulates expression of the ToxR regulon in Vibrio cholerae

TPO1, a Member of a Novel Protein Family, Is Developmentally Regulated in Cultured Oligodendrocytes

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