I'm trying to replicate the results of the DNA labeling and single molecule imaging section but have not had luck getting the same results as the publication. I was wondering if someone who has done this protocol or a similar optical mapping protocol could share some tips?
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Hi Michael,
It depends on which results of the Zhang et al. publication are proving difficult. I've had luck with a similar optical mapping method from David Schwartz's lab: Jo et al. A single-molecule barcoding system using nanoslits for DNA analysis. Proc Natl Acad Sci U S A 2007;104:2673–2678. I followed the Schwartz DNA labeling protocol precisely and it worked great. To break the visualization method down I'd suggest a simpler protocol. T7, T4, or lambda DNA can be visualized after YOYO staining by simply sandwiching the DNA in agarose between a slide and a coverslip. Squeezing the sandwich with half-melted agarose will stretch some of the DNA molecules, and you can view with a standard fluorescence microscope. Add 1-2% beta-mercapto-ethanol to reduce photobleaching. This is a "quick and dirty" method, and molecules won't be stretched uniformly, but for me it was sufficient to see that I was getting several nick-translated labeling sites on most molecules. Once you know you can make 2-color labeled DNA molecules, you could move on to better optical mapping methods with surface deposition or nanofluidics, which should lead to uniformly stretched straight molecules.
Good luck!
Brian
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