Imaging Fluorescently Stained DNA with CCD Technology How to Increase Sensitivity and Reduce Integration Times

Abstract

Ethidium bromide has long been the method of choice for staining DNA and RNA in agarose gels. However, this dye has the disadvantages of being mutagenic and toxic. To improve user safety, companies have developed alternative fluorescent dyes such as the SYBR® range of dyes1, 2 which offer a better safety profile3. The complaint of many researchers is that in the case of the SYBR® Safe stain it does not offer the same level of sensitivity as ethidium bromide and with SYBR® Green stain and SYBR® Gold stain can require longer integration times for visualisation, when viewed with UV light. UV transilluminators commonly used to excite SYBR dyes, emit UV in a broad band (approximately 100nm wide) centred at 300nm. As most fluorescent dyes have a bimodal excitation spectrum, with one excitation peak in the UV and another in the visible range, using a blue converter screen to change UV to light with an excitation range of 410-510nm, where SYBR dyes have a second excitation peak, is said to improve the sensitivity of many SYBR dyes. In this article we describe how using CCD technology with a blue converter screen and the correct lighting and filter conditions enables scientists to rapidly image SYBR Green and SYBR Gold stains with better levels of sensitivity than DNA stained with ethidium bromide, thus offering a safe yet accurate imaging method.