2008
DOI: 10.2144/000113002
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A Simple and Optimized Method of Producing Silanized Surfaces for FISH and Replication Mapping on Combed DNA Fibers

Abstract: Molecular combing of DNA is an extremely powerful DNA fiber-stretching technique that is often used in DNA replication and genome stability studies. Optimal DNA combing results mainly depend on the quality of the silanized surfaces onto which fibers are stretched. Here we describe an improved method of liquid-phase silanization using trimethoxy-octenylsilane/n-heptane as novel silane/solvent combination. Our simple method produces homogenously modified coverslips in a reproducible manner but does not require a… Show more

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Cited by 55 publications
(44 citation statements)
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“…L1 immobilization on the silicon dioxide surface and iridium oxide electrode pads were carried out as previously described with minor modifications [77,99]. Briefly, probes were cleaned and functionalized with either HNO3 (Sigma Aldrich) or by serial washes in acetone, 50% (v/v) MeOH/ H2O, and chloroform before oxygen plasma cleaning (30W) for 1 min (Harrick Plasma, PDC-001) [102]. Probes were silanized by immersion in 2% (3-mercaptopropyl) trimethoxysilane (Sigma Aldrich) solution with 4-maleimidobutyric acid N-hydroxysuccinimide ester (2 mM, Sigma Aldrich) for 1 h. Finally, probes were fully immersed in a 100 (µg/ml solution of purified L1 protein (purified at our lab) for 1 h at 4 °C, and stored in sterile 1 × phosphate buffer solution (Sigma Aldrich) until implantation.…”
Section: Methodsmentioning
confidence: 99%
“…L1 immobilization on the silicon dioxide surface and iridium oxide electrode pads were carried out as previously described with minor modifications [77,99]. Briefly, probes were cleaned and functionalized with either HNO3 (Sigma Aldrich) or by serial washes in acetone, 50% (v/v) MeOH/ H2O, and chloroform before oxygen plasma cleaning (30W) for 1 min (Harrick Plasma, PDC-001) [102]. Probes were silanized by immersion in 2% (3-mercaptopropyl) trimethoxysilane (Sigma Aldrich) solution with 4-maleimidobutyric acid N-hydroxysuccinimide ester (2 mM, Sigma Aldrich) for 1 h. Finally, probes were fully immersed in a 100 (µg/ml solution of purified L1 protein (purified at our lab) for 1 h at 4 °C, and stored in sterile 1 × phosphate buffer solution (Sigma Aldrich) until implantation.…”
Section: Methodsmentioning
confidence: 99%
“…Silanized coverslips were prepared as described before (Labit et al 2008). Thirty microliters of replicated DNA solution was pipetted onto a silanized coverslip, covered with a nonsilanized coverslip, and incubated for 5 min at RT.…”
Section: Analysis Of Replication On Single Dna Fibersmentioning
confidence: 99%
“…The quality of silanized coverslips is critical for the subsequent analysis of replication profiles. Silanized coverslips can be prepared as described [40] or purchased from the Genomic Vision company (www.genomicvision.com). The DNA solution is carefully transferred into a 2-3 ml Teflon reservoir and a silanized coverslip is incubated into the DNA solution for 5 min at room temperature.…”
Section: Melting Of Genomic Dna Plugsmentioning
confidence: 99%