title: High throughput preparation of fly genomic DNA in 96-well format using a paint-shaker.
abstract: Sample homogenization is an essential step for genomic DNA extraction, with multiple downstream applications in Molecular Biology. Genotyping hundreds or thousands of samples requires an automation of this homogenization step, and high throughput homogenizer equipment currently costs 7000 euros or more. We present an apparatus for homogenization of individual Drosophila adult flies in 96-well micro-titer dishes, which was built from a small portable paint-shaker (F5 portable paint-shaker, Ushake). Single flies are disrupted in each well that contains extraction buffer and a 4-mm metal ball. Our apparatus can hold up to five 96-well micro-titer plates. Construction of the homogenizer apparatus takes about 3-4 days, and all equipment can be obtained from a home improvement store. The total material cost is approximately 700 euros including the paint-shaker. We tested the performance of our apparatus using the ZR-96 Quick-gDNA™ kit (Zymo Research) homogenization buffer and achieved nearly complete tissue homogenization after 15 minutes of shaking. PCR tests did not detect any cross contamination between samples of neighboring wells. We obtained on average 138 ng of genomic DNA per fly, and DNA quality was adequate for standard PCR applications. In principle, our tissue homogenizer can be used for isolation of DNA suitable for library production and high throughput genotyping by Multiplexed Shotgun Genotyping (MSG), as well as RNA isolation from single flies. The sample adapter can also hold and shake other items, such as centrifuge tubes (15-50 mL) or small bottles.
authors.affiliation: a Institut Jacques Monod; CNRS; UMR 7592; Universite Paris Diderot ; Sorbonne Paris , France.
authors.forename: Michael
authors.initials: M
authors.lastname: Lang
authors.affiliation: a Institut Jacques Monod; CNRS; UMR 7592; Universite Paris Diderot ; Sorbonne Paris , France.
authors.forename: Olga
authors.initials: O
authors.lastname: Nagy
authors.affiliation: b Grootmoor 137 ; Hamburg , Germany.
authors.forename: Claus
authors.initials: C
authors.lastname: Lang
authors.affiliation: a Institut Jacques Monod; CNRS; UMR 7592; Universite Paris Diderot ; Sorbonne Paris , France.
authors.forename: Virginie
authors.initials: V
authors.lastname: Orgogozo
mesh_terms: D000818:Animals; D004247:DNA / Q000302:isolation & purification*; D004330:Drosophila / Q000235:genetics*; D005821:Genetic Techniques / Q000295:instrumentation*; D060005:Genotyping Techniques
chemical_list: D004247:DNA
keywords: 96-well format; DNA extraction; PCR; genotyping; high throughput; homogenizer; paint shaker; sample homogenization
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