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High throughput preparation of fly genomic DNA in 96-well format using a paint-shaker.

Lang M, Nagy O, Lang C, Orgogozo V - Fly (Austin) (2015)

Bottom Line: PCR tests did not detect any cross contamination between samples of neighboring wells.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.

View Article: PubMed Central - PubMed

Affiliation: a Institut Jacques Monod; CNRS; UMR 7592; Universite Paris Diderot ; Sorbonne Paris , France.

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.

No MeSH data available.


Related in: MedlinePlus

Pictures of the step-by-step construction of the homogenizer. (A-C) Pictures of the paint-shaker at 3 successive stages during the building process: (A) Paint Shaker F5 (Ushake) without the plate adapter, (B) with the horizontal clamp and (C) with the 2 vertical clamps assembled, and parts of the horizontal clamp on the right side on the table. (D) Pictures of the different parts of the plate adapter (See Fig. 5 for details). Scale bar is 5 cm.
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f0003: Pictures of the step-by-step construction of the homogenizer. (A-C) Pictures of the paint-shaker at 3 successive stages during the building process: (A) Paint Shaker F5 (Ushake) without the plate adapter, (B) with the horizontal clamp and (C) with the 2 vertical clamps assembled, and parts of the horizontal clamp on the right side on the table. (D) Pictures of the different parts of the plate adapter (See Fig. 5 for details). Scale bar is 5 cm.

Mentions: The F5 Paint Shaker (Ushake) has an adjustable horizontal clamp that can fit a wide variety of containers both in size and shape. However, the clamp had to be modified so that it could hold 96-well micro-titer plates securely in place during shaking (Fig. 3A-C).Figure 3.


High throughput preparation of fly genomic DNA in 96-well format using a paint-shaker.

Lang M, Nagy O, Lang C, Orgogozo V - Fly (Austin) (2015)

Pictures of the step-by-step construction of the homogenizer. (A-C) Pictures of the paint-shaker at 3 successive stages during the building process: (A) Paint Shaker F5 (Ushake) without the plate adapter, (B) with the horizontal clamp and (C) with the 2 vertical clamps assembled, and parts of the horizontal clamp on the right side on the table. (D) Pictures of the different parts of the plate adapter (See Fig. 5 for details). Scale bar is 5 cm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4862422&req=5

f0003: Pictures of the step-by-step construction of the homogenizer. (A-C) Pictures of the paint-shaker at 3 successive stages during the building process: (A) Paint Shaker F5 (Ushake) without the plate adapter, (B) with the horizontal clamp and (C) with the 2 vertical clamps assembled, and parts of the horizontal clamp on the right side on the table. (D) Pictures of the different parts of the plate adapter (See Fig. 5 for details). Scale bar is 5 cm.
Mentions: The F5 Paint Shaker (Ushake) has an adjustable horizontal clamp that can fit a wide variety of containers both in size and shape. However, the clamp had to be modified so that it could hold 96-well micro-titer plates securely in place during shaking (Fig. 3A-C).Figure 3.

Bottom Line: PCR tests did not detect any cross contamination between samples of neighboring wells.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.

View Article: PubMed Central - PubMed

Affiliation: a Institut Jacques Monod; CNRS; UMR 7592; Universite Paris Diderot ; Sorbonne Paris , France.

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.

No MeSH data available.


Related in: MedlinePlus