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Biotechniques. 2003 Jun;34(6):1238-43.

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

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1
Department Biologie I, Bereich Biodiversitätsforschung: Systematische Botanik Ludwig-Maximilians-Universität Menzinger Str. 67, 80638 München, Germany. meimberg@botanik.biologie.uni-muenchen.de

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.

PMID:
12813891
[Indexed for MEDLINE]
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