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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

Quality control and cross contamination test. (A) Genomic DNA extracted with our homogenizer. M: 1 kb DNA Ladder (NEB) 0.5 μg/lane. (B) Schematic drawing of the arrangement of the samples in the 96-well plate. 1–13 indicate wells containing single fly specimen, −1 and −2 indicate wells devoid of flies. (C) PCR amplification products of GAL4 inserts. PCR products are of expected size. 1: VT054796-GAL4 2275 bp, 2: VT054833-GAL4 2270 bp, 3: GMR15E09-GAL4 4029 bp, 4: VT054820-GAL4 2219 bp, 5: VT054795-GAL4 2188 bp, 6: VT054841-GAL4 2184 bp, 7: VT054829-GAL4 2288 bp, 8: GMR15E09-GAL4 4029 bp, 9: VT054824-GAL4 2202 bp, 10: VT054838-GAL4 2177 bp, 11: GMR13B12-GAL4 3586 bp, 12: VT054839-GAL4 2212 bp, 13: GMR13B12-GAL4 3586 bp. M: 1 kb DNA Ladder (NEB) 0.5 μg/lane.
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f0002: Quality control and cross contamination test. (A) Genomic DNA extracted with our homogenizer. M: 1 kb DNA Ladder (NEB) 0.5 μg/lane. (B) Schematic drawing of the arrangement of the samples in the 96-well plate. 1–13 indicate wells containing single fly specimen, −1 and −2 indicate wells devoid of flies. (C) PCR amplification products of GAL4 inserts. PCR products are of expected size. 1: VT054796-GAL4 2275 bp, 2: VT054833-GAL4 2270 bp, 3: GMR15E09-GAL4 4029 bp, 4: VT054820-GAL4 2219 bp, 5: VT054795-GAL4 2188 bp, 6: VT054841-GAL4 2184 bp, 7: VT054829-GAL4 2288 bp, 8: GMR15E09-GAL4 4029 bp, 9: VT054824-GAL4 2202 bp, 10: VT054838-GAL4 2177 bp, 11: GMR13B12-GAL4 3586 bp, 12: VT054839-GAL4 2212 bp, 13: GMR13B12-GAL4 3586 bp. M: 1 kb DNA Ladder (NEB) 0.5 μg/lane.

Mentions: We pooled ten single fly extractions and examined the size distribution of genomic DNA fragments on an agarose gel (Fig. 2A). We observed that genomic DNA was partially degraded into fragments that were smaller than 10 kb but that a substantial fraction of DNA was larger than 10 kb.Figure 2.


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)

Quality control and cross contamination test. (A) Genomic DNA extracted with our homogenizer. M: 1 kb DNA Ladder (NEB) 0.5 μg/lane. (B) Schematic drawing of the arrangement of the samples in the 96-well plate. 1–13 indicate wells containing single fly specimen, −1 and −2 indicate wells devoid of flies. (C) PCR amplification products of GAL4 inserts. PCR products are of expected size. 1: VT054796-GAL4 2275 bp, 2: VT054833-GAL4 2270 bp, 3: GMR15E09-GAL4 4029 bp, 4: VT054820-GAL4 2219 bp, 5: VT054795-GAL4 2188 bp, 6: VT054841-GAL4 2184 bp, 7: VT054829-GAL4 2288 bp, 8: GMR15E09-GAL4 4029 bp, 9: VT054824-GAL4 2202 bp, 10: VT054838-GAL4 2177 bp, 11: GMR13B12-GAL4 3586 bp, 12: VT054839-GAL4 2212 bp, 13: GMR13B12-GAL4 3586 bp. M: 1 kb DNA Ladder (NEB) 0.5 μg/lane.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f0002: Quality control and cross contamination test. (A) Genomic DNA extracted with our homogenizer. M: 1 kb DNA Ladder (NEB) 0.5 μg/lane. (B) Schematic drawing of the arrangement of the samples in the 96-well plate. 1–13 indicate wells containing single fly specimen, −1 and −2 indicate wells devoid of flies. (C) PCR amplification products of GAL4 inserts. PCR products are of expected size. 1: VT054796-GAL4 2275 bp, 2: VT054833-GAL4 2270 bp, 3: GMR15E09-GAL4 4029 bp, 4: VT054820-GAL4 2219 bp, 5: VT054795-GAL4 2188 bp, 6: VT054841-GAL4 2184 bp, 7: VT054829-GAL4 2288 bp, 8: GMR15E09-GAL4 4029 bp, 9: VT054824-GAL4 2202 bp, 10: VT054838-GAL4 2177 bp, 11: GMR13B12-GAL4 3586 bp, 12: VT054839-GAL4 2212 bp, 13: GMR13B12-GAL4 3586 bp. M: 1 kb DNA Ladder (NEB) 0.5 μg/lane.
Mentions: We pooled ten single fly extractions and examined the size distribution of genomic DNA fragments on an agarose gel (Fig. 2A). We observed that genomic DNA was partially degraded into fragments that were smaller than 10 kb but that a substantial fraction of DNA was larger than 10 kb.Figure 2.

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