Selection of chloroplasts by laser microbeam microdissection for single-chloroplast PCR

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Abstract

Laser microbeam microdissection and laser pressure catapulting offer the possibility of separating cell compartments, thus allowing for contamination-free analysis. Using these methods, we were able to select single chloroplasts of Nicotiana tabacum. Starting from homogenized leaf material, chloroplasts were purified by differential centrifugation and applied directly onto a poly-ethylene-naphthalate membrane that was mounted on a microscope slide. Single chloroplasts were dissected under microscopic control and catapulted into a PCR tube. Subsequent PCR of a spacer region between the trnT and trnF genes verified the successful amplification of DNA from a single chloroplast. The advantage of this method compared to the use of capillaries or optical tweezers is that one is able to prepare high numbers of samples in a short time.

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Meimberg, H., Thalhammer, S., Brachmann, A., Müller, B., Eichacker, L. A., Heckl, W. M., & Heubl, G. (2003). Selection of chloroplasts by laser microbeam microdissection for single-chloroplast PCR. BioTechniques, 34(6), 1238–1243. https://doi.org/10.2144/03346rr01

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