0.1 mm zirconium beads

Manufactured by Biospec

Sourced in France, United States, United Kingdom

The 0.1 mm zirconium beads are small, spherical particles made of zirconium. They are primarily used for mechanical disruption and homogenization of samples in various laboratory procedures.

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Lab products found in correlation

15 protocols using 0.1 mm zirconium beads

Bacterial DNA Extraction from Mucosal Biopsies

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Three pieces of mucosal tissue were obtained from patients with TA, SSA/P, or CRC by endoscopic biopsy. Additionally, four biopsy samples were taken from adjacent normal tissues of each patient.
Extraction of bacterial DNA was performed from mucosal biopsy samples as we previously reported25 (link). Briefly, 100 mg of frozen gastric mucosal tissues was suspended in 750 μL of sterile bacterial lysis buffer (200 mmol/L NaCl, 100 mmol/L EDTA [pH 8.0], 20 mmol/L Tris base, 20 mg/mL lysozyme) and incubated at 37 °C for 30 minutes. Then, we added 20 μL of proteinase K and 80 μL of 10% SDS to the mixture and incubated it at 65 °C for 30 minutes. Finally, bead beating was performed for 90 seconds at 5,300 rpm (PRECELLYS 24; Bertin Technologies, Le Bretonneux, France) after adding a 300 mg of 0.1-mm zirconium beads (BioSpec Products, Bartlesville, OK, USA) for finishing homogenization. The homogenized mixture was cooled on ice and then centrifuged at 14,000 rpm for 5 minutes. Bacterial DNA was extracted from the supernatant by phenol/chloroform/iso-amyl alcohol (25:24:1) followed by chloroform/iso-amyl alcohol (24:1), and precipitated by absolute ethanol at −20 °C for 1 hour. The precipitated DNA was suspended in DNase-free H₂O and cleaned up using a DNA clean-up kit according to the manufacturer’s instructions. Isolated DNA was stored at −80 °C until use in microbial characterization

Park C.H., Han D.S., Oh Y.H., Lee A.R., Lee Y.R, & Eun C.S. (2016). Role of Fusobacteria in the serrated pathway of colorectal carcinogenesis. Scientific Reports, 6, 25271.

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Gastric Mucosal DNA Extraction Protocol

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The methods of DNA extraction from mucosal biopsy samples were described previously^{5 (link),9 (link)}. Briefly, 100 mg of frozen gastric samples was suspended in 750 μL sterile bacterial lysis buffer (200 mmol/L sodium chloride, 100 mmol/L ethylenediaminetetraacetic acid, 20 mmol/L Tris base, and 20 mg/mL lysozyme) and incubated at 37 °C for 30 min. Then, we added 20 μL proteinase K and 80 μL 10% sodium dodecyl sulfate to the mixture, and incubated it at 65 °C for 30 min. Bead beating was performed for 90 s at 6.9 g (PRECELLYS 24; Bertin Technologies, Le Bretonneux, France) following adding 300 mg of 0.1 mm zirconium beads (BioSpec Products, Bartlesville, OK, USA) to complete the homogenization. The homogenized mixture was cooled on ice and centrifuged at 18.3 g for 5 min. DNA was extracted from the supernatant using phenol/chloroform/isoamyl alcohol (25:24:1), then chloroform/isoamyl alcohol (24:1) and followed by precipitated with absolute ethanol at −20 °C for 1 h. The precipitated DNA was suspended in DNase-free H₂O and cleaned using a DNA clean-up kit (QIAGEN, Hilden, Germany). We stored isolated DNA at −80 °C until microbial characterization.

Park C.H., Lee J.G., Lee A.R., Eun C.S, & Han D.S. (2019). Network construction of gastric microbiome and organization of microbial modules associated with gastric carcinogenesis. Scientific Reports, 9, 12444.

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Bacterial DNA Extraction from Mucosal Biopsies

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Extraction of bacterial DNA from mucosal biopsy samples was performed as previously described.12 Briefly, 100 mg of frozen gastric mucosal tissues was suspended in 750 μL of sterile bacterial lysis buffer (200 mM NaCl, 100 mM EDTA [pH 8.0], 20 mM Tris base, 20 mg/mL lysozyme) and incubated at 37°C for 30 minutes. Next, 20 μL of proteinase K and 80 μL of 10% SDS were added to the mixture followed by incubation at 65°C for 30 minutes. Finally, bead beating was performed for 90 seconds at 6.9 g (PRECELLYS 24; Bertin Technologies, Le Bretonneux, France) after adding 300 mg of 0.1‐mm zirconium beads (BioSpec Products, Bartlesville, OK) to finish homogenization. The homogenized mixture was cooled on ice and then centrifuged at 18.3 g for 5 minutes. Bacterial DNA was extracted from the supernatant by phenol/chloroform/iso‐amyl alcohol (25:24:1) followed by chloroform/iso‐amyl alcohol (24:1) and precipitated by absolute ethanol at −20°C for 1 hour. The precipitated DNA was suspended in DNase‐free H₂O and cleaned using a DNA clean‐up kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Isolated DNA was stored at −80°C until microbial characterization.

Park C.H., Lee A., Lee Y., Eun C.S., Lee S.K, & Han D.S. (2018). Evaluation of gastric microbiome and metagenomic function in patients with intestinal metaplasia using 16S rRNA gene sequencing. Helicobacter, 24(1), e12547.

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Genomic DNA Extraction from Water Samples

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To isolate genomic DNA from the 279 water samples (93 days in triplicates) we used a bead beating approach. Filters were sterilely cut and placed into 2 mL screw-cap tubes, followed by the addition of 0.25 g of sterile 0.1 mm zirconium beads (BioSpec Products Inc., Bartlesville, OK). Cells were lysed by adding 750 μL of Cell Lysis Solution (Qiagen, USA) and shaken in a Mini Beadbeater-1 (BioSpec Products, Inc., Bartlesville, OK) at 5000 rpm for 60 s, followed by incubation at 80 °C for 5 min. Once samples were cooled down to room temperature, RNA was digested by adding 4 µL RNAse A (4 mg/mL), mixed by inverting the tubes, and incubated at 37 °C for 30 min. DNA was purified by adding 250 µL of Protein Precipitation Solution (Qiagen, USA), mixed by vortexing for 20 s, incubated on ice for 5 min and centrifuged at 13,000 rpm for 5 min. Supernatant was transferred to a new tube and centrifuged again to ensure removal of all precipitates. Subsequently, 750 µL of isopropanol were added to 750 µL of supernatant to precipitate DNA, and incubated at −20 °C overnight. DNA was recovered by centrifugation at 13,000 rpm for 5 min, and washed with 700 µL of 70% ethanol. Finally, the DNA was dried and resuspended in 100 µL of DNA hydration solution (Qiagen, USA).

Martin-Platero A.M., Cleary B., Kauffman K., Preheim S.P., McGillicuddy D.J., Alm E.J, & Polz M.F. (2018). High resolution time series reveals cohesive but short-lived communities in coastal plankton. Nature Communications, 9, 266.

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Saliva DNA Extraction with Zirconium Beads

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DNA was extracted from 100 µl of raw saliva and 200 µl of culture-enriched samples using a modified mag forensics extraction kit protocol (LGC Genomics, Berlin, Germany). All samples were thawed on ice, vigorously vortexed for 20 seconds then transferred to 1.5 ml Eppendorf tubes containing 650 µl 0.1 mm zirconium beads (Biospec Products, Bartlesville, OK) in lysis buffer and 550 µl phenol (Sigma-Aldrich, St Louis, MO). Samples were mechanically disrupted through two cycles of 2 minutes bead beating (Mini-Beadbeater-24, Biospec Products) and 2 minutes on ice, before centrifugation at 12,000×g for 2 minutes. The aqueous DNA phase was then transferred to new Eppendorf tubes pre-filled with 10 µl of magnetic particle suspension in 1.3 ml binding buffer. Following 30 minutes of incubation in a shaker at room temperature, the beads were washed with 200 µl wash buffers 1 and 2, and then dried for 20 minutes at 55°C. DNA was eluted in 50 µl template volumes and stored at +4°C. The procedure was repeated when more template was required.

Wyllie A.L., Chu M.L., Schellens M.H., van Engelsdorp Gastelaars J., Jansen M.D., van der Ende A., Bogaert D., Sanders E.A, & Trzciński K. (2014). Streptococcus pneumoniae in Saliva of Dutch Primary School Children. PLoS ONE, 9(7), e102045.

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Fecal DNA Extraction from Mice

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Fecal pellets were collected from mice and stored at -80°C. Fecal DNA was extracted using a phenol-chloroform extraction protocol described by the CBDM Laboratory (Harvard Medical School, Boston, MA). Briefly, frozen fecal specimens were incubated at room temperature for 2 hours with 500 μl of 0.1 mm zirconium beads (BioSpec Products) in 710 μl Lysis Buffer containing 100mM NaCl, 10mM Tris, 100mM EDTA, 0.2 mg/ml Proteinase K and supplemented with DNAse-free 5.9% SDS (Fisher Scientific) solution. 500 μl phenol:chloroform:isoamyl alcohol (24:24:1) reagent was added to each sample followed by cell disruption for 2 minutes using Mini bead-beater (Biospec Products). Then, samples were centrifuged (5,900 x g, 3 min, 4°C) and the aqueous phase was transferred into a new tube and again extracted with 300 μl of phenol:chloroform:isoamyl alcohol. After centrifugation (15,700 x g, 3 min, 4°C) the aqueous phase was collected into a new tube and DNA was precipitated with equal volume of (-20°C) isopropyl alcohol and 1:10 (v/v) 3M sodium acetate. Samples were incubated on ice, pelleted by centrifugation (15,700 x g, 20 min, 4°C), rinsed with cold ethanol, spun and dried. Purified DNA was resuspended in 200 μl TE buffer (Amresco) and quantified on Nanodrop ND-1000. DNA samples were stored at -80°C.

Laubitz D., Harrison C.A., Midura-Kiela M.T., Ramalingam R., Larmonier C.B., Chase J.H., Caporaso J.G., Besselsen D.G., Ghishan F.K, & Kiela P.R. (2016). Reduced Epithelial Na+/H+ Exchange Drives Gut Microbial Dysbiosis and Promotes Inflammatory Response in T Cell-Mediated Murine Colitis. PLoS ONE, 11(4), e0152044.

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Bacterial DNA Extraction for 454-Sequencing

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For 454-sequencing, bacterial DNA was purified using a modified QIAGEN protocol. In brief, bacteria from 1,900 μl FVU were harvested by centrifugation at 30,000×g for 15 min, re-suspended in 200 μl of 30 mM Tris/HCl pH 8.0; 1 mM EDTA, 15 mg/ml lysozyme, 20μg/ml protease, supplemented with 200 μl AL buffer (QIAGEN, Hilden, Germany) and subjected to bead-beating (0.1 mm zirconium beads, Biospec Products, Bartletsville, OK, USA) at speed setting 7000 for 70s in a MagNA Lyser (Roche). This was followed by 10 min incubation at 56 °C and purification using the DNeasy blood and tissue kit as recommended by the manufacturer (QIAGEN). The DNA was eluted in 200 μl 10 mM Tris/HCl pH 8.5 and stored at –20 °C until 16S PCR for the 454-sequencing was performed.
For the species specific qPCRs, DNA was extracted by boiling the cell pellet with Chelex-100 resin as previously described [20 ].

Frølund M., Wikström A., Lidbrink P., Abu Al-Soud W., Larsen N., Harder C.B., Sørensen S.J., Jensen J.S, & Ahrens P. (2018). The bacterial microbiota in first-void urine from men with and without idiopathic urethritis. PLoS ONE, 13(7), e0201380.

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Stool DNA Extraction Protocol

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Stool specimens were handled according to the GEMS protocol (16 (link)). DNA was isolated from frozen stool specimens by using a bead beater with 3-mm diameter solid glass beads (Sigma-Aldrich, St. Louis, MO, USA) and, subsequently, with 0.1-mm zirconium beads (BioSpec Products, Inc., Bartlesville, OK, USA) to disrupt cells. The cell slurry was then centrifuged at 16,000 × g for 1 min, and the supernatant was processed by using QIAamp DNA Stool Extraction Kit (QIAGEN, Valencia, CA, USA). Extracted DNA was precipitated with ethanol and shipped to the United States.

Lindsay B., Oundo J., Hossain M.A., Antonio M., Tamboura B., Walker A.W., Paulson J.N., Parkhill J., Omore R., Faruque A.S., Das S.K., Ikumapayi U.N., Adeyemi M., Sanogo D., Saha D., Sow S., Farag T.H., Nasrin D., Li S., Panchalingam S., Levine M.M., Kotloff K., Magder L.S., Hungerford L., Sommerfelt H., Pop M., Nataro J.P, & Stine O.C. (2015). Microbiota That Affect Risk for Shigellosis in Children in Low-Income Countries. Emerging Infectious Diseases, 21(2), 242-250.

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Gastric Mucosal DNA Extraction Protocol

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The DNA was extracted from the gastric mucosal tissue as previously described [12 (link),13 (link)]. Briefly, 100 mg frozen gastric mucosal tissues was suspended in 750 μL sterile bacterial lysis buffer (200 mmol/L sodium chloride [NaCl], 100 mmol/L ethylenediaminetetraacetic acid [EDTA, pH 8.0], 20 mmol/L Tris base, and 20 mg/mL lysozyme). After incubation at 37°C for 30 minutes, 20 μL proteinase K and 80 μL 10% sodium dodecyl sulfate (SDS) were added to the mixture. Then, it was incubated at 65°C for 30 minutes. Finally, bead beating was performed for 90 seconds at 6.9 g (PRECELLYS 24; Bertin Technologies, Le Bretonneux, France) following adding 300 mg of 0.1 mm zirconium beads (BioSpec Products, Bartlesville, OK, USA) to complete the homogenization. The homogenized mixture was cooled on ice, followed by centrifuging at 18.3 g for 5 minutes. DNA was extracted from the supernatant using phenol/chloroform/isoamyl alcohol (25:24:1) followed by chloroform/isoamyl alcohol (24:1). Then it was precipitated with absolute ethanol at -20°C for 1 hour. The DNA was suspended in DNase-free H₂O and cleaned using a DNA clean-up kit (QIAGEN, Hilden, Germany). Isolated DNA was stored at -80°C until the microbial characterization.

Choi S., Lee J.G., Lee A.R., Eun C.S., Han D.S, & Park C.H. (2019). Helicobacter pylori antibody and pepsinogen testing for predicting gastric microbiome abundance. PLoS ONE, 14(12), e0225961.

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Optimized Stool DNA Extraction

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DNA was extracted and purified with either the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany; for samples from S1 to S3) or QIAamp Fast DNA Stool Mini Kit (for samples from S4 to S6) according to kit availability and with the following modifications: A total of 200–250 mg processed stool was added to tubes containing 300 mg of 0.1 mm zirconium beads (BioSpec Products, Inc. Bartlesville, OK, USA) suspended in 100 μl lysis buffer (200 mM of NaCl, 100 mM of Tris-HCl, 20 mM of EDTA and 20 mg/ml of lysozyme). After 30 min incubation with 500 rpm mixing at 37°C, the ASL buffer from the QIAamp DNA Stool Mini Kit (or InhibitEX buffer from QIAamp Fast DNA Stool Mini Kit) was added into the sample mixture for homogenization with two cycles on a MP FastPrep-24 instrument (MP Bio MP Biomedicals LLC, Santa Ana, CA, USA) at 6.5 m/s for 1 min. The supernatant was further processed using QIAamp kits according to the manufacturer’s instructions and the purified DNA was stored at -20°C until PCR amplification.

Hsieh Y.H., Peterson C.M., Raggio A., Keenan M.J., Martin R.J., Ravussin E, & Marco M.L. (2016). Impact of Different Fecal Processing Methods on Assessments of Bacterial Diversity in the Human Intestine. Frontiers in Microbiology, 7, 1643.

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