Daniel:Notebook/HiResChrPaint/2013-12-9

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FISH Research[edit]

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I have been doing research today on various FISH methods/buffers used. Below are some of my results:

  • Formamide, for example, lowers the melting temperature of DNA, allowing for hybridization to occur at lower temperatures. However, increased formamide concentrations lead to more specific probe binding (called stringent hybridization solutions). I might not want these stringent wash conditions, as my analysis in to probe sequence has indicated that my probes probably have plenty of errors in them. Therefore, decreasing formamide concentration (30-40%) might be the best way to go.
  • Washing post-hybridization. The steps post hybridization are very important for how much signal you want to wash away. Higher formamide concentrations (I use 0%) are considered more stringent. Also, low salt concentrations (I use 2X SSCT and 60C for the first wash, 2X SSCT at RT for the second, and 0.2X SSC at RT for the last) are considered more stringent. High temperature is also considered more stringent of a wash method.