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 any sophisticated or expensive equipment in comparison to other known silanization protocols. However DNA fibers were combed onto these coverslips with very good high-density alignment and stayed irreversibly bound onto the surfaces after various denaturing treatments, as required for different immunofluorescent detection of DNA with incorporated modified nucleotides or FISH.

Neuroadhesive L1 coating attenuates acute microglial attachment to neural electrodes as revealed by live two-photon microscopy

“…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.…”

Sperm is epigenetically programmed to regulate gene transcription in embryos

“…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.…”

Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing

“…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.…”