| Popis: |
Accurate and quick determination of DNA concentration is critical for the assembly of synthetic constructs, as well as a multitude of other experiments. We sought to optimize an under-utilized and inexpensive approach for determining DNA concentration: a spotting technique that uses the intercalating dye Ethidium Bromide. This technique does not require specialized equipment such as a spectrophotometer, but instead relies on visualization of dye-DNA complex fluorescence when excited by UV light. We modelled and tested a range of parameters for dye concentration and spot size, finding that 15uL spots with 1.0ug/mL Ethidium Bromide produced the most reliable standard curve. More importantly, we hope that our approach can help other labs optimize this protocol for their own experimental setup. Adoption of this technique may help enable development of iGEM teams in resource limited environments and laboratories which do not or cannot employ a satisfactory method for determining DNA concentration.Financial DisclosureWe obtained funding support from Canon Solutions America, and indirect support for this work through the CUNY Research Scholars Program. However, the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing InterestsThe authors have declared that no competing interests exist.Ethics StatementN/AData AvailabilityYes – all data are fully available without restriction. The data can be found in the associated supplementary materials.Article TagsDNA, Ethidium Bromide, EtBr, Concentration, Method, UV, Gibson AssemblyHeader StatementThis work was assessed during the iGEM/PLOS Realtime Peer Review Jamboree on 23rd February 2018 and has been revised in response to the reviewers. Responses to Reviewers, and supplementary information (including Fig. S1) are available as separate files. |
