MP Biomedicals FastPrep® Family

Michael Hemkemeyer, Geertje J. Pronk, Katja Heister, Ingrid Kogel-Knabner, Rainer Martens & Christoph C. Tebbe.

FEMS Microbiol Ecol.2014. Vol 90.

• Key words: Artificial soils; soil minerals; soil particle size fractions; soil microbial diversity; terminal restriction fragment length polymorphism
• Aim of the study: Analyse the importance of different mineral compositions for the diversity of soil microorganisms application quantitative PCR
• Application: Artificial soil
• Material: FastPrep-24™ instrument, FastDNA™ Spin Kit for Soil containing Lysing Matrix E
• Buffer: Sodium Phosphate buffer and MT buffer supplied with the FastDNA™ Spin Kit for Soil

DNA was extracted with the FastDNA™ Spin Kit for Soil using the FastPrep-24™ instrument according to the manufacturer’s instructions, using following modifications:

1. Volumes of sodium phosphate buffer and supplied "MT-buffer" were adjusted to 950 and 120 µL, respectively.
2. Bead-beating was run twice for 45 s at a speed of 6.5 m/s.
3. The samples were then centrifuged for 5 min at 14,000 x g and room temperature.
4. The DNA bound to the binding matrix of the FastDNA™ Spin Kit was washed twice with 1 mL 5.5 M guanidinthiocyanate to remove coextracted contaminants.
5. After elution of DNA with 100 µL distilled water, this step was repeated using the eluate.

• The combination of FastDNA™ Spin Kit for Soil and the FastPrep-24™ instrument provide a high quality of gDNA.
• The qPCR results revealed that the mineral composition and the particle size fractions have specific and different selective effects on soil samples.

Orin C. Shanks, Catherine A. Kelty, Mano Sivaganesan, Manju Varma, and Richard A. Haugland

Appl and Envir. Microbiol. 2009. Vol 75.

• Key words: Waterborne disease, environmental waters, microbial community, DNA extraction
• Aim of the study: Development of a method to assess microbial community present in waste water sample
• Quantitive PCR: Quantitative PCR
• Sample Name: Wastewater
• Material: FastPrep-24™ instrument, FastDNA™ Spin Kit for Soil containing Lysing Matrix E
• Buffer: Sodium Phosphate Buffer and MT Buffer supplied with the FastDNA™ Spin Kit for Soil

500ml of primary effluent was collected and immediately stored on ice. 25 ml of each sample was filtered through a 0.2-µmpore size supor-200 filters and each filter was placed in a sterile 1.5 ml microtube and stored at -80°C. For DNA extraction.

1. Cut the freezed filters with a sterile cutter.
2. Add the cutted filters to a Lysing Matrix E tubes.
3. Add 978 µl of Sodium Phosphate Buffer and 122 µl of MT buffer, provided with the FastDNA™ spin kit for Soil.
4. Homogenize in the FastPrep-24™ instrument for 120 seconds at a speed setting of 6.0
5. Centrifuge at 14,000 x g for 5-10 minutes to pellet debris.
6. Proceed with the FastDNA™ Spin Kit for Soil extraction protocol.

• The FastPrep-24™ and associated matrices have demonstrated successful lysis and DNA extraction from 20 samples of wastewater in only 120 seconds.
• This method saves hours of work during sample preparation, ensures high purified DNA and an effective PCR amplification.

Takada Y, Matsumoto N.
Japan Agricultural Research Quartorly. 2005. Vol.39.

The challenge with extractions from soil is isolating DNA or RNA without contamination by humic acids or other PCR inhibitors. Effective, efficient sample preparation is critical to succcessful downstream results.
DNA extraction from Andisol, a volcanic ash soil, is known to be very difficult because this soil has a complex matrix, including allophane as a clay mineral. Soil properties such as high clay content contribute to high adsorption of DNA to soil particles.

• Key words: Environmental DNA, microbial community analysis, molecular methods, unculturable microorganisms.
• Aim of the study: Improvement of DNA extraction from volcanic ash soil
• Application: PCR
• Sample Name: Andisol
• Sample Type: Volcanic ash soil
• Material: FastPrep-24™ instrument, FastDNA™ Spin Kit for Soil, skim milk (carrier minimizing adsorption of nucleic acids to soil)

• DNA could successfully be extracted from Andisol soil samples with the FastDNA™ Spin Kit for Soil and the addition of 40 mg of skim milk per gram of soil sample. PCR products of the expected size were amplified from all extracts with skim milk.
• Resultant extracts were suitable for PCR and no other purification procedures were needed.

Jörg D. Ettenauer, Guadalupe Piñar, Ksenija Lopandic, Bernhard Spangl, Güther Ellersdorfer, Christian Voitl, Katja Sterflinger

Science of the Total Environment. 2012. Vol 439.

• Key words: Building materials, DNA extraction, DNA purity, PCR
• Aim of the study: Analyzing microbial communities in building materials
• Application: Plaster, red brick and gypsum
• Material: FastPrep-24™ homogenizer, FastDNA™ Spin Kit for Soil containing Lysing Matrix E
• Buffer: Sodium Phosphate Buffer & MT Buffer (provided with the Kit)

1. Sampling was done using a sterile scalpel or an ethanol flamed hammer and a chisel to remove the material from the walls and collected it in sterile plastic bags.
2. The transport and storage of the sampling material were done at room temperature.
3. In the laboratory, samples were ground for 2 min in liquid nitrogen using a sterile mortar and pestle, collected in a sterile 50 ml falcon tube and homogenized by manual shaking.
4. Three different sample amounts of each material, 50 mg, 100 mg and 250 mg (each in triplicate), were weighed in a Sartorius precision scale for each extraction method.
5. Tubes were either immediately processed or stored at -20°C. The resulting nine samples for each method were further subjected to the different DNA extraction methods.
6. When using the FastDNA™ Spin Kit for Soil method, samples were processed two times in the FastPrep® homogenizer for 40 s at a speed of 6 m/s.

• Up to thirteen extraction methods were evaluated with three building materials. The FastDNA™ Spin kit for soil showed to be the best DNA extraction method and could provide positive results for all tests with all tested samples.
• This study shows that the FastPrep® extraction method is a gold standard for quantification of indoor fungi and bacteria in building materials.

Carlos Calderon-Vasquez, Mary L. Durbin, Vanessa E.T.M Ashworth, Livia Tommasini Kapua K.T Meyer, and Michael T. Clegg

J. Amer. Soc. Hort. Sci. 2013. Vol 138 (4).

• Key words: Persea Americana, molecular markers, vitamins, tocopherol, sitosterol, breeding.
• Aim of the study: Heritability estimation of carotenoids, β-sitosterol and α-tocopherol content in ripe avocado fruits.
• Application: Spectrophotometric assay
• Sample type: Avocado mesocarp tissue
• Material: FastPrep-24™ instrument, 2 ml Lysing Matrix D tubes
• Buffer: 1 ml of absolute ethanol, 6% pyrogallol and 5 ml HCl

1. 0.5 g of avocado mesocarp tissue were transferred to a 2 ml lysing matrix D tubes
2. 1 ml of absolute ethanol, 6% pyrogallol and 5 ml HCl have been added to the tubes
3. The tubes were set up in the FastPrep-24™ instrument
4. The homogenate was incubated for 15 min at 70°C and then left to cool down to room temperature with addition of 20 μL of a 0.4% NaCl solution
5. Extraction of the homogenate was done with 4 ml hexane/petroleum ether (1:1)

• The compounds were successfully extracted from the avocado samples allowing their identification and quantification
• The FastPrep® extraction method in combination with Lysing Matrix D tubes could also be used or adapted to extract DNA from other species.

Gang-hua Lang, Mika Ohba, Shinichi Kawamoto, Koichi Yoza, Tatsuya Moriyama and Kazumi Kitta

Food Sci. Technol. Res. 2010. Vol 16.

• Key words: Rice (Oryza sativa), RAG2, α-amylase/trypsin inhibitor, recombinant protein, polyclonal antisera, processed food.
• Aim of the study: Allergen identification in processed food.
• Application: SDS-PAGE analysis
• Sample type: Cereals
• Material: FastPrep-24™ instrument, 2 ml Lysing Matrix D tubes, Lab Mixer
• Buffer: Buffer A (30 mM Tris-HCl, pH 8.0 supplemented with 1 M NaCl)

1. Grains of various cereals were crushed into flour with a Lab Mixer
2. 100 mg of grain flour was transferred to a 2 ml Lysing Matrix D tubes
3. 1 ml of Buffer A was added to the tubes
4. After brief vortexing, the tubes were incubated on ice for 1 h
5. The tubes were then set up in a FastPrep® System at speed 6.0 for 40 sec
6. After centrifugation at 15,000 x g, 4°C for 10 min, the supernatant was dispensed into several tubes and stored at -30°C

• The use of FastPrep® system in combination with lysing matrix D allowed isolation and characterization of the main allergens in rice grains.

Christophe Monnet, Vincent Ulve, Anne-Sophie Sarthou, and Françoise Irlinger

APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 2006. Vol 72.

• Key words: Cheese, Microbial flora, L.Lactis, RNA analysis, RT-PCR
• Aim of the study: Understanding the cheese microbial flora without separating the cells from the cheese matrix.
• Application: Quantification of L.Lactis rRNA and mRNA by real-time PCR
• Sample name: Cheese
• Material: FastPrep® Homogenizer, 2 ml Lysing Matrix tubes containing 0.1 mm silica/ zirconium beads
• Buffer: TRIzol reagent

1. Approximately 125 mg of cheese was placed into a 2 ml lysing matrix tubes.
2. 1.25 ml of TRIzol reagent was added immediately.
3. The tubes were vigorously shaken in a bead beater (FastPrep-24™ system) by using three 60 s mixing sequences at a speed of 6.5 m/s. The tubes were cooled on ice for 5 min before each mixing sequence.
4. After centrifugation for 10 min at 12,000 x g and 4°C, each supernatant (approximately 1,100 μl) was transferred into a 2-ml tube containing 300 μl of a gel that improved separation of the aqueous and organic phases (Phase Lock Gel Heavy; Eppendorf, Hamburg, Germany).

• In the present work, RNA was successfully extracted from cheeses manufactured with L. lactis, and rRNA and mRNA transcripts were quantified by real-time PCR.
• The FastPrep® extraction method could be used or adapted for cheeses in which other microbial species are present.

Carlos Infante, Aniela Crespo, Eugenia Zuasti, Marian Ponce, Laura Pérez, Victoria Funes, Gaetano Catanese, Manuel Manchado.

Food Research International. 2006. Vol 39.

• Key words: S. scombrus, ND5, multiplex-PCR, canned products, authentication
• Aim of the study: Authentification of Atlantic mackerel in canned products
• Application: Multiplex-PCR
• Sample name: Atlantic mackerel Scomber scombrus
• Sample type: Fish
• Material: FastPrep-24™ instrument, FastDNA™ Kit containing 2 ml Lysing Matrix A tubes
• Buffer: Cell Lysis Solution-TC provided with the FastDNA™ Kit

1. Add 150 mg of raw or canned muscle sections in 2 ml lysing matrix A tubes
2. Add 1 ml of CLS-TC buffer
3. Load tubes in FastPrep-24™ instrument and process 40 sec at speed setting of 5.0
4. Centrifuge to pellet debris
5. Performe DNA isolation following FastDNA™ Kit's protocol

• A multiplex-PCR assay for the authentication of the Atlantic mackerel Scomber scombrus in commercial canned products has been developed using FastPrep-24™ instrument for homogenization and FastDNA™ Kit for purification.
• The system was assayed using six different canned products labeled as S.scombrus. Positive identification in all samples was observed, revealing this methodology as a potential molecular tool for direct application in the authentication of S. scombrus canned products.

Ibrahim Mehmeti, Fadime Kiran, and Ozlem Osmanagaoglu

Afr. J. Biotechnol. 2011. Vol 10.

• Key words: FastPrep®, Sonication, centrifugation, lactic acid bacteria (LAB)
• Aim of the study: Development of an optimized protein extraction protocol
• Application: One dimensional (1D) SDS-PAGE. Two dimensional (2D)-PAGE
• Sample type: Bacteria (E. faecalis, P. pentosaceus and L. lactis)
• Material: Sonicator, centrifuge, FastPrep® 120 instrument
• Buffer: 8M Urea, 2M Thiourea, 0,5% CHAPS, 10 mM DTT and 0,1% immobilized pH gradient (IPG)

1. Pellets were resuspended in 400 μL rehydration buffer containing 9M Urea, 2M Thiourea, 0,5% CHAPS, 10 mM DTT and 0,1% immobilized pH gradient (IPG).
2. Cells were lysed with acid washed glass beads (diameter of 212 to 300 μm) using a FastPrep® 120 at speed 6 m/s for
   3 x 45 sec. at 4°C. After each cycle the solution was kept on ice for 1 min.
3. Cell debris were removed by centrifugation at 14,000 x g for 10 min at 4°C.
4. Before analysis, the supernatant was kept at -20°C.

• The results show that higher amount of proteins were obtained when the cells were lysed with a FastPrep®. Six times higher protein concentration, which is important for proteomics, was obtained with extraction by FastPrep®.
• SDS-PAGE gel images show that higher amounts of proteins are obtained only when proteins extracted by FastPrep® method.
• More than 400 protein, spots, with isoelectric points (pI) ranging from 4.0 to 7.0 and molecular weights (MW) from 0 to 100 kDa, were observed with 2D-PAGE analysis. In addition, proteins with less abundance and high molecular weights were resolved clearly and detected strongly on 2D gel when FastPrep® method was used.
• FastPrep® extraction method was an efficient and reliable method for lysing and/or extracting proteins of LAB for proteomic approach and reproducible amounts of bacterial proteins can successfully be extracted.
• Pictures were excellent enough to be used in alignment for statistical analyses and spots well-resolved for MALDI TOF analyses.

Alexander Rohde, Jens Andre Hammerl, Bernd Appel, Ralf Dieckmann, and Sascha Al Dahouk.

BioMed Research International. 2015.

• Key words: Food microbiology, pathogen identification, sampling, homogenization
• Aim of the study: Identification of an efficient food homogenization method for microbiological testing
• Sample type: Chicken breast, salami and meat paste
• Material: FastPrep-24™, Bagmixer 400, SpeedMill, Branson Sonifier 450, 6.35 mm ceramic beads, 50 ml tubes
• Buffer: 1% buffered peptone water

1. Samples were diluted 1: 10 in buffered peptone water (1.37 ml for FastPrep® homogenization)
2. Samples were placed in sterile Falcon 50 ml tubes filled with three 6.35 mm ceramic beads
3. Tubes were loaded in the FastPrep-24™ and processed at speed 5m/s for 30, 1 min, 2, 4 and 8 mins
4. Aliquots were diluted appropriately in buffered peptone water and plated twice in suitable dilution steps on selective medium
5. Colonies were enumerated after an incubation period of 24 h at 37°C

• The results of this study show that the FastPrep® homogenization method gives the best results with high reproducibility for detection of surface food contamination.
• For inner-matrix contamination, long treatments are required and only FastPrep-24™, as a large-volume homogenizer, produced consistently good recovery rates, extracting seven times more pathogen after 8 mins than stomaching.

Kyoungwon Cho, Kwang-Soo Cho, Hwang-Bae Sohn, In Jin Ha, Su-Young Hong, Hyerim Lee, Young-Mi Kim and Myung Hee

Journal of Experimental Botany. 2016. Vol. 67.

• Key words: Anthocyanin, metabolomics, colored potato, RNA-seq, ultrapressure liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS)
• Aim of the study:Understanding of regulatory networks related to anthocyanin biosynthesis and identification of key regulatory genes
• Application: UPLC-Q-TOF-MS, RNA sequencing
• Sample type: Three potato cultivars (light-red Hongyoung, dark-purple Jayoung and white Atlantic)
• Material: FastPrep-24™ instrument

1. 200 mg of frozen potato sprouts were ground in the FastPrep-24™ instrument
2. Samples has been processed 3 times at speed of 4.5m/s for 25 sec
3. Mixed sprouts were suspended in 600 μL methanol and a 0.125% formic acid solution
4. The solution was kept at 4°C for 30 min and then sonicated at 4°C for 20 sec by using three repetitions at 20kHz and 250W
5. The solution was then centrifuged at 3000 rpm for 15 min at 4°C
6. The supernatant solution was centrifuged during 10 min at 13 000 rpm and at 4°C

• FastPrep® is the technology of choice for metabolites extraction and characterization. In the present work, metabolites were successfully extracted from potato samples allowing their profiling.

Andy V. Roberts.

Cytometry Part A. 2007. Vol 71.

• Key words: Bead beating, fresh leaves, herbarium leaves, petals, pollen, Allium cepa, Nicotiana tabacum, Petroselinum crispum, Rosa canina, Rosa rugosa
• Aim of the study: Use of a bead beating method to prepare suspensions of plant nuclei for measurement of DNA amou nts by flow cytometry.
• Application: Nuclei staining and DNA measurement by flow cytometry
• Sample name: Rosa canina, Rosa rugosa, Nicotiana tabacum
• Sample type: Leaves, Petals & Pollen
• Material:FastPrep-24™ instrument, 2 ml Lysing Matrix tubes containing 1mm silica beads or 2.5 mm Zirconia/silica beads
• Buffer: Lysis buffer contained 0.1 M citric acid and 0.5% Triton X-100 in deionized distilled water

1. First, 7 mg of fresh leaf tissue was placed in a 2 ml screw capped tube along with 10 zirconia/silica beads (2.5 mm diameter) and 1 ml of lysis buffer, and homogenized at speed of 5 m/s for 45 s with the FastPrep-24™ instrument.
2. The suspension was then filtered and the filtrate was added to 4 mg of pollen in a fresh tube, along with glass beads of 1.0 mm diameter.
3. Lysis buffer was added to bring the total volume to 1 ml.
4. The mixture of leaf and pollen nuclei was then homogenized at 4 m/s for 30 s with the FastPrep™ instrument.
5. The final suspension was then filtered and the filtrate was incubated with RNase at 37°C for 30 min.

• Bead beating with the FastPrep-24™ instrument enabled suspensions of nuclei to be prepared simultaneously from 12 samples by agitating for 45 s. In a normal working day, the author was able to prepare, stain, and analyze suspensions of up to 36 leaf samples and estimate DNA amounts in three replicates per sample by flow cytometry.
• In the standard method, the gloved hand of the experimenter is in contact with harmful substances in the nuclei isolation buffer. The more remote handing of the isolation buffer in the bead beating method reduces this potential hazard.
• The versatility of the bead beating method was demonstrated with suspensions of nuclei prepared from fresh leaves of species that range in DNA amounts from 1.13 pg (R. rugosa) to 33.5 pg (A. cepa), suspensions of nuclei from fresh petals and dry of herbarium leaves of R. canina, and pollen of R. rugosa.

FastPrep® and associated matrices have demonstrated successful lysis and RNA extraction from cassava roots in only 60 seconds.

Because plant samples can be very fibrous and contain high levels of polyphenolic compounds, polysaccharides, and RNases,it can be extremely difficult to extract enough usable DNA or RNA for PCR analysis and other downstream applications. The FastDNA™ Kit, FastDNA™ Spin Kit, and FastRNA™ Pro Green Kit make it fast and simple!
Effective, efficient sample preparation is critical to successful downstream results.

• Case study: Root
• Key words: RNA extraction, roots, fibrous plants
• Aim of the study: Optimization of RNA extraction from fibrous roots
• Application: RT-qPCR
• Sample name: Cassava Root
• Sample type: Root
• Material: FastPrep-24™ instrument, Lysing Matrix A with an additional 1/4 inch ceramic bead

1. Add the Cassava root sample to a Lysing Matrix A tube containing an additional 1/4 inch ceramic bead.
2. Add 1 ml of RNApro extraction buffer
3. Homogenize in the FastPrep-24™ instrument for 60 sec at a speed setting of 6.0.
4. Centrifuge at 14,000 x g for 5 - 10 minutes to pellet debris.
5. Collect the supernatant and proceed with the RNA extraction protocol.

• RNA could successfully be extracted from cassava roots with the FastPrep System. Total RNA yields achieved were up to 110 μg of RNA per μl.

Stéphanie M. Swarbreck, Erika A. Lindquist, David D. Ackerly, and Gary L. Andersen.

Plant Cell Physiol. 2011. Vol 52.

• Key words: Avena barbata, climate change, ESTs, root
• Aim of the study: Generation of a large amount of cDNA sequence data for transcriptomic studies in A. barbata.
• Application: Transcriptome analysis by Sanger sequencing & pyrosequencing
• Sample name: Avena barbata
• Sample type: Root
• Material:FastPrep-24™ Homogenizer
• Buffer: Modified CTAB (CetylTrimethylAmmonium Bromide) buffer: 50 ml of 0.1 M of aluminum ammonium sulfate and 0.5 ml of phenol: chloroform: isoamyl alcohol (25: 24: 1)

1. 0.5 ml of modified CTAB buffer was added to the samples
2. Samples were bead beaten for 30 s at 5.5 m/s in a FastPrep-24™ instrument
3. Samples were centrifuged at 16,000 x g for 5 min at 4°C.
4. A second extraction with the modified CTAB buffer was conducted
5. A 1 ml aliquot of chloroform was then added to the aqueous supernatant followed by a centrifugation at 12,000 x g for 5 min at 4°C.
6. 2 vols. of 30% (w/v) polyethylene glycol 6,000 in 1.6 M NaCl solution and 1 ml of linear acrylamide were added to the aqueous supernatant to precipitate the nucleic acids.
7. The RNA/DNA pellet was subsequently washed with 60% ice-cold ethanol and resuspended in diethylpyrocarbonate (DEPC)-treated water.

• The results show that the FastPrep-24™ extraction method generates a good-quality RNA for sequencing
• The combined use of pyrosequencing and Sanger sequencing was successful in generating a high number of expressed sequence tags (ESTs).

Jean-Marc Routaboul, Christian Dubos, Gilles Beck, Catherine Marquis, Przemyslaw Bidzinski, Olivier Loudet and Loic Lepiniec.
Journal of Experimental Botany. 2012. Vol 63

• Key words: Arabidopsis, flavonoids, metabolite profiling, natural variation, quantitative trait loci
• Aim of the study: Identification of genes controlling flavonoid metabolism
• Application: Flavonoid extraction and analysis by LC-MS
• Sample name: Arabidopsis
• Sample type: Seeds
• Material:FastPrep-24™ homogenizer
• Buffer: Acetonitrile/Water (3/1; v/v) or Methanol/Acetone/water/Trifluoroacetic acid (30/42/28/0.05; v/v/v/v)

1. Three representative seed aliquots from the three biological repeats were pooled before flavonoid extraction.
2. All seed samples were ground for 90 s at maximum speed with a FastPrep-24™ homogenizer in 1 ml of solvent mixe.
3. A 4 μg aliquot of apigenin was added as an internal standard.
4. Following centrifugation, the pellet was extracted further with 1 ml of the same solvent mixes overnight at 4°C.

• The use of FastPrep-24™ system succeeded in full homogenization of seeds, allowing flavonoid extraction, quantification and complete analysis of their metabolism by LC-MS method.

Dr. Loïc Faye, ANGANY Genetics, France

• Key words: Protein expression, plant screening, recombinant protein
• Aim of the study: Selection of the best producers of a recombinant protein used for allergic patients desensitization
• Application: Western Blot analysis
• Sample name: Leaves
• Material:FastPrep-24™ homogenizer, 2 ml Lysing Matrix D tubes containing 1.4 mm ceramic spheres
• Buffer: Laemmli denaturing sample buffer containing 60 mM Tris-Cl pH 6.8, 1% SDS, 10% glycerol, 2% beta-mercaptoethanol

1. Snap one tobacco leaf disc (10 mg) in a Lysing Matrix D tube
2. Add 200 μl of denaturing buffer to the Lysing Matrix D tube
3. Homogenize the plant tissue with the FastPrep-24™ instrument for 60 seconds at a speed setting of 4.0 m/s
4. Transfer supernatant from the Lysing Matrix D tube into an Eppendorf tube
5. Boil each sample for 5 min and then centrifuge 1 min at 12.000 rpm to pellet the cellular debris
6. Load 15 μl of each supernatant in a 15% polyacrylamide gel for protein separation by electrophoresis

• The FastPrep® system is a powerful tool to get rapidly and with a very high reproducibility protein extracts ready for electrophoresis (SDS-PAGE) and Western Blot analysis.
• The FastPrep-24™ has also been successfully used for protein extraction from other plants (Arabidopsis, lettuce etc...) in the same conditions using less than 15 mg of plant material for sample preparation.
• Similar experiments have been performed for protein extraction in non-denaturing conditions using ice cold buffer (100 mM Tris buffer, pH 7.4 containing 10% sucrose, 5 mM EDTA, 0.28% b-mercaptoethanol, 5 ml PMSF and 0.3 ml aprotinin) and plant samples stored at -70°C in lysing matrix D tubes before extraction.
• The process is then scaled-up by using the BigPrepTM adapter for homogenization in 2 x 50 ml tubes. Two large protein samples are prepared simultaneously from 1-2 g of plant tissues in 50 ml lysing Matrix D tubes.

S. Wesley Long, Randall J. Olsen, Shrenik C. Mehta, Timothy Palzkill, Patricia L. Cernoch, Katherine K. Perez, William L. Musick, Adriana E. Rosato, James M. Musser.

Antimicrobial Agents and Chemotherapy. 2014. Vol 58.

• Key words: Genome sequencing, antibiotic resistance, clinical isolates, ceftaroline
• Aim of the study: Understanding antibiotic resistance mechanism in clinical isolates of Staphylococcus aureus.
• Application: Genome sequencing
• Sample Name: Patient expectorated sputum & blood
• Material: FastPrep-96™ Instrument, Lysing Matrix B tubes
• Buffer: Tryptic soy broth

1. Patient isolates were grown on tryptic soy agar supplemented with 5% sheep blood.
2. Five of the isolates grew from expectorated sputum. And the sixth isolate was obtained from an aerobic blood culture bottle.
3. Genomic DNA was isolated from multiple colonies grown overnight in tryptic soy broth.
4. The cells were lysed using Lysing Matrix B in a in a FastPrep-96™ instrument.

• The use of the high-throughput FastPrep-96™ homogenizer in combination with Lysing Matrix B tubes allows high quality DNA extraction and genome sequencing analysis of ceftaroline-resistant methicillin-resistant Staphylococcus aureus (MRSA).
• Genome sequencing results confirm a previously undescribed high-level antibiotic resistance mechanism in clinical isolates of MRSA.

• Key words: Protein extraction, gram-positive bacteria, restriction enzyme
• Aim of the study: Demonstrate the capacity of the FastPrep® System to deal with otherwise difficult to lyse bacteria.
• Application: Restriction enzymes extraction
• Sample Name:Bacillus amyloliquefaciens and Staphylococcus aureus 3A
• Material: FastPrep-24™ FastProtein™ Blue Kit containing Lysing Matrix B tubes

1. Cell density.
• Sonication: Bacterial suspensions of 0.2 g wet weight (w/w) and 0.15 g (w/w) per ml of buffer for Bacillus amyloliquefaciens and Staphylococcus aureus 3A, respectively.
2. Disruption.
   • Sonication: Bacteria are disrupted at 50% maximum intensity (large tip) for Bacillus amyloliquefaciens and 20% maximum intensity (small tip) for Staphylococcus aureus 3A with a Branson Sonicator B30. Temperature is maintained at 4°C 5°C by cooling in an ice salt water bath. Sonication was continued for 10 min in 40 sec. bursts for Bacillus amyloliquefaciens and 60 sec. in 5 sec. bursts for Staphylococcus aureus 3A.
   • FastPrep®: The FastProtein™ Blue matrix was used. Tubes containing the lysing matrix and samples were prechilled at 4°C then mixed. Samples are homogenized with the FastPrep-24™ instrument at speed 6.0 for 40 sec. for Bacillus amyloliquefaciens and at speeds 4.0 and 6.0 for 20 sec. and 40 sec. respectively for Staphylococcus aureus 3A. The tubes were returned to the ice bath. Homogenization and chilling was repeated for all time points.

At each time point a 50 μl sample was taken, centrifuged for 5 min at 4°C in a benchtop centrifuge and tested for OD260 and activity.

These experiments clearly show that the FastPrep-24™ instrument using FastProtein™ Blue matrix can be used to successfully extract unstable enzymes from gram positive bacteria. Even in cases where sonication can release active materials (such as the Bacillus amyloliquefaciens experiments here), the lysing time can be reduced by approximately 60%. For samples like Staphylococcus aureus 3A that require longer and less efficient methods of lysis (such as French Press), the FastPrep® method offers clear advantages for extraction of active proteins.

• Key words: Mouse bone samples, RNA extraction, FastPrep-24™ homogenizer, gene expression
• Aim of the study: High quality RNA extraction from mouse bone samples
• Application: qPCR
• Sample Name: Mouse Calvaria, long bones, chondrocytes, Osteoblasts (OB).
• Material: FastPrep-24™ homogenizer, Lysing Matrix S (metal beads), Lysing Matrix D (ceramic beads)
• Buffer: RNA extraction buffer

1. Put bone samples immediately in liquid nitrogen after sampling
2. Place bone sample in Lysing Matrix tube containing 6 metal beads (Lysing Matrix S)
3. Add 350 μl of RNA extraction buffer per sample
4. Load tube in the FastPrep-24™ homogenizer and process 2 x 15 sec at speed setting of 6m/s with 5 min intermediate incubation on ice
5. Centrifuge Lysing Matrix tube at 12 000 x g, 5 min at 4°C
6. Transfer supernatant to a new 2 ml microcentrifuge tube and follow RNA extraction according to RNA extraction protocol

• Bone sample grinding is challenging. This study shows that the use of FastPrep-24™ homogenizer in combination with metal beads is highly performant for this application.
• High RNA quality and yield are extracted from mineralized Osteoblasts and Chondrocytes using FastPrep-24™, Lysing Matrix D & Lysing Matrix S tubes.
• FastPrep® technology is the most adapted for bone & Forensics studies.

Allergy is a hypersensitivity disorder of the immune system. According to epidemiological studies, at present, 20-30% of the population in many countries around the world suffers from allergies, and this percentage is growing trend.

Allergens are proteins with a broad range of molecular weights (5-50 kDa) exhibiting different features of solubility and stability, able to cause IgE-mediated hypersensitivity after contact with the immune system.

The development of new types of allergy treatments needs diverse and well-characterized allergenic source materials. This study describes an effective method for allergen characterization.

• Key words: Allergy, citrus, allergen isolation, citrus homogenization
• Aim of the study: identification of grinding method for citrus allergen isolation
• Application: West blot analysis
• Sample Type: Citrus
• Sample Name: Green lemon, yellow lemon, orange, grapefruit
• Material: FastPrep-24™ 5G, CoolPrep adapter, 2ml Lysing Matrix A, C & E tubes, IKA grinder
• Buffer: PBS

1. Add citrus peel and pulp with 500μl of PBS in 2 ml Lysing Matrix A, C or E tubes.
2. Load Lysing Matrix tubes in a CoolPrep Adapter containing dry ice.
3. Process with the FastPrep-24™ 5G: 40 sec at a speed setting of 6.0 m/s.
4. Centrifuge the Lysing Matrix tubes 20 min at 18.000 x g, 4°C to pellet debris.
5. Keep the supernatant at -20°C prior to analyses.

• Protein extraction from peel and pulp of citrus samples with the FastPrep-24™ 5G showed to be highly effective with the 3 types of lysing matrix tested.
• The protein yield with IKA grinder method was very low with high concentration of pectins, preventing protein migration on SDS- PAGE gel.
• The effectiveness of the FastPrep® method is quantitative, higher protein yield, and qualitative, wide variety composition of protein extracts pectins free.
• The FastPrep® system is a powerful tool to get rapidly and with a very high reproducibility protein extracts ready for electrophoresis (SDS-PAGE) analysis.
• Protein extracted with the FastPrep-24™ 5G instrument have conserved their immunoreactivity

• Key words: Food-borne disease, Parasites identification, Human feces, qPCR, K.septempunctata
• Aim of the study: Identification of a standard method for DNA extraction from fecal parasitesn
• Application: Quantitative PCR
• Sample Name: Human fecal sample
• Material:FastDNA™ Spin Kit for Soil containing Lysing Matrix E (MP Biomedicals), QIAamp DNA stool minikit (Qiagen), UltraClean fecal DNA kit (Mo Bio)
• Buffer: provided with each of the three commercial DNA extraction kits

To compare the amount of K.septempunctata (parasites) DNA extracted using the three kits.

1. 200 mg of each sample and 200 μl of DNA elution buffer were used during the extraction procedure for each kit.
2. Extracted DNA was stored at -20°C until use.

• The FastDNA™ Spin Kit for Soil showed to be the best DNA extraction method providing the highest PCR amplification.
• The FastPrep® technology gives higher yields and increases detection limit threshold of PCR. FastDNA™ Spin Kit for Soil is the most efficient method for extracting parasites DNA from fecal samples.

• Key words: DNA extraction, mice feces, mice cecal content, 16S rDNA, qPCR
• Aim of the study: Selection of an optimal DNA extraction method for molecular assays
• Application: Quantitative PCR
• Sample Name: Mice feces and intestinal contents
• Material: FastDNA™ Spin Kit for Soil, FastDNA™ Spin Kit for Feces, QIAam™ DNA stool minikit, MasterPure™ Gram Positive DNA Purification Kit, NucliSENS™ easyMAG, ZR Fecal DNA MiniPrep™. FastPrep-24™ Instrument
• Buffer: Buffers provided with each DNA extraction kit

1. Feces were pooled and frozen at -20°C immediately after collection.
2. Cecal samples were obtained shortly after dissection and immediately frozen in liquid nitrogen and stored at -80°C before use.
3. With each extraction method tested, DNAs were extracted from 50 mg of starting material (wet weight) in five duplicates.
4. For three bead beating methods: FastDNA™ Spin Kit for Soil, FastDNA™ Spin Kit for Feces and ZR Fecal DNA MiniPrep™, DNA extraction was performed with the FastPrep-24™ homogenizer at speed 6 m/s for 40s.

• Among seven DNA extraction methods, The FastDNA™ Spin Kit for Soil showed to be the most efficient extraction method for both feces and intestinal contents providing the highest DNA yield and 16S rDNA.
• DNA fragments recovered were larger than 1.6 kb making them suitable for PCR-analysis of microbiomes.
• This study shows that the FastPrep® technology (FastPrep® homogenizer and FastDNA™ Spin Kit for Soil) is adapted for detecting genes of various Gram-positive bacteria present in fecal and cecal matrices.

Allergy is a hypersensitivity disorder of the immune system. According to epidemiological studies, at present, 20-30% of the population in many countries around the world suffers from allergies, and this percentage is growing trend.

Pollen are significant sources of clinically relevant out door aeroallergens, recognized as both a major trigger for, and cause of, allergic respiratory diseases.

Allergens are proteins with a broad range of molecular weights (5-50 kDa) exhibiting different features of solubility and stability, able to cause IgE-mediated hypersensitivity after contact with the immune system. The development of new types of allergy treatments needs diverse and well-characterized allergenic source materials. This study describes an effective method for allergen characterization.

• Key words:Allergen, IgE immunoreactivity, pollen homogenization, hypersensitivity community, DNA extraction
• Aim of the study: identification of fast method for protein extraction from pollen grains
• Application: Western blot analysis
• Sample Name: Birch, Nettle, Wall Pellitory pollens
• Sample Type: Pollen
• Material: FastPrep-24™ 5G instrument, CoolPrep adapter, 2 ml Lysing Matrix C & E tubes
  
• Buffer: PBS

1. Incubation Method
   • Add 50 mg of pollen and 500 μl of PBS in a tube
   • Place the tube in a shaker for 18 hours, in cold room
   • Centrifuge the suspension 20 mins at 18 000 x g, 4°C
   • Keep the supernatant at -20°C prior to analyses
2. Grinding method
• Add 50 mg of pollen and 500μl of PBS in 2 ml Lysing Matrix C or E tube.
• Load Lysing Matrix tubes in a CoolPrep Adapter, containing dry ice.
• Process with the FastPrep-24 5G: 40 sec at a speed setting of 6.0 m/s.
• Centrifuge the Lysing Matrix tubes 20 mins at 18 000 x g, 4°C to pellet debris.
• Keep the supernatant at -20°C prior to analyses

• Protein extraction from pollen samples with the FastPrep-24™ 5G showed to be highly effective compared to the standard method based on overnight incubation.
• The effectiveness of the FastPrep® method is quantitative, higher protein yield, and qualitative, wide variety composition of protein extracts.
• The FastPrep® system is a powerful tool to get rapidly and with a very high reproducibility protein extracts ready for electrophoresis (SDS-PAGE) analysis.
• IgE immunoreactivity is conserved in protein extracted with the FastPrep-24™ 5G instrument.

Skin tissue, although easily accessible, is difficult to process owing to its natural resistance to mechanical shearing and high levels of RNases and proteases. Currently, these complications result in degraded RNA samples with variable yield.

We have developed a method of sequential extraction of high quality RNA and protein from a single 3 mm full thickness skin punch biopsy.

• Key words:Tissue biopsy, clinical samples, RNA extraction, protein isolation
• Aim of the study: Optimization of RNA and Protein extraction from skin tissue
• Application: Western blot & quality RNA analysis
• Sample Name: Tissue biopsy
• Sample Type: Human skin biopsies from a 3 mm punch
• Material: FastPrep-24™ instrument, Lysing matrix D tubes
  
• Buffer: Guanidine thiocyanate lysis buffer

1. Add the 19 mg of skin sample to a Lysing Matrix D tube
2. Add 1 ml of a guanidine thiocyanate lysis buffer (5.1M guanidine thiocyanate, 50 mM sodium citrate, 50 mM EDTA, 0.5% β-mercaptoethanol).
3. Homogenize in the FastPrep-24™ instrument for 3 x 40 seconds at a speed setting of 6.0. Place the tubes on ice for 5 mins between each run.
4. Centrifuge at 14,000 x g for 5-10 mins to pellet debris
5. Proceed with the RNA and protein extraction protocol

• Sample variability and exposure to exogenous contamination were reduced using the FastPrep® bead beating, this instrument allowed processing up to 24 samples very quickly.
• This method yields 1-2 mg of RNA and 150 mg of protein, which is usable in many sensitive downstream applications including microarray, quantitative real-time PCR, two-dimensional gel electrophoresis and Western blot analysis.

• Key words:Tissue biopsy, medical microbiology, bacterial identification
• Aim of the study: Isolation of microorganisms from infected biopsy specimens
• Application: Bacterial culture
• Sample Name: Tissue biopsy
• Material: FastPrep-24™ 5G Homogenizer, TeenPrep sample holder, 8-10 ceramic beads (Lysing Matrix M, 1/4")
• Buffer: glucose broth

Tissue biopsies are transferred with tweezers from original glass and into 15 ml sterile centrifuge tube (use 2 ml tube if very small biopsy).

1. Add between 1 g and 2 g biopsy sample to a 15 ml Lysing Matrix tube containing 8-10 large 1/4 inch ceramic beads
2. Add 5ml glucose broth
3. Homogenize samples in the FastPrep-24™ 5G instrument for 40 seconds at a speed setting of 6.5 m/s.
4. After homogenizing, take out the tube(s) from the FastPrep-24™ 5G and remove the beads.
5. Inoculate media and transfer the homogenized biopsy to a sterile glass for storage

• The FastPrep® System (FastPrep-24™ 5G, TeenPrep sample holder, Lysing Matrix M) provides effective homogenization of tissue biopsies and did not result in cross-contamination of the samples.
• The culture results were proven better than the manual method.

• Key words: DNA extraction kits, PCR- DGGE analysis, fecal specimens.
• Aim of the study: Extraction of bacterial genomic DNA from human fecal specimens
• Application: PCR-DGGE
• Sample Name: Human fecal specimens
• Material: Mobio Ultra Clean Fecal DNA extraction kit (M), QIAamp DNA Stool Mini Kit (Q), FastDNA™ Spin Kit (FSp), FastDNA&term; Spin Kit for Soil (FSo), FastPrep-24™ instrument, Vortex
  
• Buffer: Provided with each DNA extraction kit

1. Wet fecal specimen weight used for extraction: 10, 25, 50, 100 and 200 mg.
2. DNA extractions were made following each manufacturer instructions.
3. For FastDNA™ Spin Kit (Fsp) and FastDNA™ Spin Kit for Soil (FSo), samples were loaded in FastPrep-24™ homogenizer and processed 40 sec at speed setting of 6 m/s.
4. For M and Q Kits, samples were loaded in FastPrep-24™ homogenizer and processed 30 sec at speed setting of 5.5 m/s.c

• Protein extraction from pollen samples with the FastPrep-24™ 5G showed to be highly effective compared to the standard method based on overnight incubation.
• The effectiveness of the FastPrep® method is quantitative, higher protein yield, and qualitative, wide variety composition of protein extracts.
• The FastPrep® system is a powerful tool to get rapidly and with a very high reproducibility protein extracts ready for electrophoresis (SDS-PAGE) analysis
• IgE immunoreactivity is conserved in protein extracted with the FastPrep-24™ 5G instrument.

Various virology institutes reported recently a new method for the isolation of intact virus particles from infected animal tissues for studies of pathogenic viruses (ex: avian Influenza A viruses, i.e H5N1) and development of vaccines. This simple and reproducible method allows to accurately measure the viral load in tissues, follow the spread of the virus in mouse organs and assess the effect of vaccination.

• Keywords: Virus isolation, influenza A virus, infected animal tissues, pathogenic viruses
• Aim of the study: Isolation of intact viruses from infected animal tissues
• Application: Virus titration
• Sample Type: Tissue
• Sample Name: Mouse lung tissue
• Material: FastPrep-24TM instrument, 2 ml lysing matrix tubes containing 1/4 inch ceramic beads
  
• Buffer: Hank's balanced salt solution containing 0.5% lactalbumin, 10% glycerol, 200 U/ml penicillin, 200 g/ml streptomycin, 100 U/ml polymyxin B sulfate, 250 μg/ml gentamycin, and 50 U/ml nystatin.

1. Snap freeze the weighed lung of a mice (100-150 mg) in a Lysing matrix M tube and store at -70°C.
2. Add 1 ml of ice-cold Buffer to the Lysing Matrix M tube.
3. Homogenize the tissue with the Fastprep-24™ Instrument for 20 seconds at a speed setting of 4.0 m/s.
4. Incubate the tube on ice for 2 minutes.
5. Homogenize the tissue a second time with the Fastprep-24™ Instrument for 20 seconds at 4.0 m/s.
6. Add 0.5 ml of medium to the Lysing Matrix tube and centrifuge 1 minute at 10.000 rpm to pellet the tissue debris.
7. Transfer the supernatant containing the virus particles to a new microcentrifuge tube.
8. Infect MDCK cells with quintuplicated 10-fold serial dilutions of the supernatants as previously described (1).
9. HA activity of the culture supernatants collected 5 days post inoculation are used as indicator of infection. Titers are calculated according to Spearman-Karber's method 3.

• The Fastprep® System together with Lysing Matrix M tubes (2 ml tubes containing one 1/4 inch ceramic bead) were successfully used to homogenize infected tissues and release intact viral particles as a first step of this experimental procedure.

• Key words: Yeast, DNA, RNA, Extraction, Capsule, Method, Cryptococcus neoformans
• Aim of the study: Development of consistent extraction method for DNA & RNA extraction from resistant strains of Cryptococcus neoformans
• Application: RNA extraction
• Sample Name: Cryptococcus neoformans
• Sample Type: Yeast
• Material: FastPrep-24TM instrument, 0.4-0.6 mm glass beads
• Buffer: PBS

1. Cells from 15 ml overnight culture (approx.10⁹) were collected, washed with cold water, resuspended in 200 μl of TE and distributed into two 1.7 ml microcentrifuge tubes.
2. 0.5 ml of glass beads (0.4-0.6 mm), 250 μl of RNA lysing solution, 250 μl 4 M guanidine thiocyanate with 25 mM sodium citrate, pH 7, 0.1 M β-mercaptoethanol, 500 μl phenol, pH 5 and 100μl chloroform/isoamyl alcohol (24 :1) were added to each tube.
3. Both tubes were placed in the FastPrep-24™ instrument and processed in 4 cycles of 40 sec each. Between the cycles samples were placed on ice.
4. After last cycle tubes were removed from the instrument, placed 5 min in ice and spun 10 min at 13,000 x g.
5. The upper phase was transferred to fresh microcentrifuge tube for RNA purification.

• RNA purification is accomplished using FastPrep® system and glass beads after a preliminary bead beating treatment
• Yields range around 1 mg RNA from 15 ml overnight culture (10⁹ cells)
• RNA appears undegraded, making it suitable for molecular manipulations

Jayne Hazon, Richard Poulton, John Sullivan, Alistair Derrick Alere Toxicology Plc. 2015.

The 2014 Society of Hair Testing (SoHT) consensus hashighlighted EtG as the first choice marker for abstinence assessment and also for proving chronic excessive alcohol consumption in child custody cases. Currently EtG samples are incubated in a sonication bath overnight to extract the analytes from the hair samples, meaning that turnaround time is significantly increased compared to other assays within the laboratory.

• Key words: Hair grinding, ETG extraction, toxicology, GCMS
• Aim of the study: Ethyl Glucuronide extraction from hair samples
• Application: LC-MSMS
• Sample Name: Hair
• Material: FastPrep-24™ instrument, steel and/or ceramic banded beads
• Buffer: Deionised water

1. Cut 75 mg of hair samples into 3-6 cm sections
2. Put the cutted hair samples into 2 ml tubes containing steel and/ or ceramic beads
3. Add deionized water
4. Load the tubes in the FastPrep-24™ instrument and process 2 x 1 min at speed setting of 6.0 m/s

• The use of a Fastprep-24™ bench homogenizer reduce the extraction time for Ethyl Glucuronide (EtG) from overnight to just 40 seconds in hair samples.
• Grinding hair samples with the FastPrep-24™ system has a clear advantage over simply cutting hair in terms of EtG recovery.
• The added cost of consumables and equipment is mitigated by the dramatic reduction in extraction time and improvement in customer service.

• Key words: Abstinence, nails, contamination, LC-MS/MS, norcocaine, cocaine, cocaethylene, benzoylecgonine
• Aim of the study: Retrospective monitoring of cocaine consumption of recreational and dependent users
• Application: Cocaine and metabolites analyses by LC-MS/MS
• Sample Name: Toenails
• Material: FastPrep-24™ Homogenizer, 2 ml Lysing Matrix S tubes containing metal beads

1. Nail clippings were washed twice with 1 ml of water and acetone, respectively, by vortexing for 30s.
2. They were cut into snippets and approximately 5 mg were weighed into Lysing Matrix Tubes containing metal beads for
   pulverization at 5.5 m/s (8-times for 60s) by a FastPrep-24™ homogenizer.

• Toenails may be an alternative for retrospective monitoring of cocaine consumption/abstinence.