HIGH THROUGHPUT NUCLEIC ACID PURIFICATION SYSTEMS

Transcription

1 HIGH THROUGHPUT NUCLEIC ACID PURIFICATION SYSTEMS nucleic acid purification utilizing magnetic beads & silica plate technologies Innovations in Nucleic Acid Isolation

2 COMPANY PROFILE Since its founding in 1998, Omega Bio-tek, Inc. has been at the forefront of nucleic acid purification by offering products for clinical and basic research, biotechnology and agricultural applications. DNA and RNA extraction is the first step for so many downstream analyses, and efficient and clean nucleic acid isolation is crucial. Our goal is to offer high quality products to help you improve your workflows. Our diverse product line ranges from RNA purification from plants to DNA extraction from dried blood spots. By offering over 9 products for nucleic acid isolation, we have a solution to almost any of your nucleic acid purification needs. Contact one of our specialists to see which product would best fit your application. With the ability to offer individual components and customize kits, we can help your lab reduce waste and increase productivity. Quality is key to operations at Omega Bio-tek. We offer products for manual and automated processing and we support our customers by having Hamilton STAR, Beckman Coulter Biomek FX and Thermo KingFisher instruments in house. In addition to offering nucleic acid purification products, we also offer next generation sequencing services utilizing Illumina platforms. We are ISO 91:28 certified and we ensure that our products are properly assembled, tested, recorded, stored and shipped. We perform rigorous quality checks of our products and thoroughly train our employees to ensure compliance. We also have several quality control steps within our processes to deliver the best product. We firmly believe that quality in equals quality out. THE OMEGA DIFFERENCE ECONOMICAL: On average, Omega Bio-tek s products cost 3% less than the competition. INDIVIDUAL COMPONENTS: We sell kit components separately. CUSTOMIZABLE: For large customers, we can generate custom packaging, kits, reagents, etc. PRODUCT RANGE: Alternative options for almost all of your nucleic acid purification needs. 1

3 MAG-BIND PARTICLES Our Mag-Bind technology provides a flexible and reliable method for isolating nucleic acids using magnetic beads. Our magnetic bead-based kits are optimized in processing times and binding capacity for each application. Magnetic beads are available in 24-, 96- or 384-well formats to fit your sample size. The same kit can be modified for different formats by changing the volumes, plasticware and magnetic stands. HIGH THROUGHPUT PRODUCTS Magnetic beads allow for higher recovery and yield of nucleic acids versus filter-based methods. Magnetic stands with SBS standard format can be added to liquid handlers, which can be specifically programmed for an inexpensive way to process multiple plates simultaneously. Mag-Bind technology is ideally suited for liquid handlers that have a 96-channel head, but it can be adapted to 8-channel head instruments. Mag-Bind kits can also be used with Thermo KingFisher Flex, Thermo KingFisher DUO, Qiagen BioSprint and Life Technologies MagMAX 96. MAG-BIND PARTICLES SILICA PLATE TECHNOLOGY Omega Bio-tek HiBind technology is the basis for our E-Z 96 silica plates that have a binding capacity of up to 5 µg of genomic DNA or total RNA and 2 µg of plasmid DNA. Our kits use semi-skirted 96-well plates that fit manual and automated vacuum manifolds and plate adaptors for centrifugation. The E-Z 96 silica plates have a silica glass fiber matrix that offers excellent well-to-well consistency and includes tip directors to eliminate cross contamination. The plates feature a 75 µl well capacity to reduce sample processing time. The E-Z 96 silica plates are an ideal option for liquid handlers with 8- or 96-channel pipettors. VACUUM MANIFOLDS The E-Z 96 vacuum manifold is designed for manual DNA/RNA sample preparation with E-Z 96 DNA/RNA plates. The E-Z vacuum manifold is also compatible with other semi-skirted filter plates. The upper plate has a molded recess that fits our filter plate and contains a custom gasket to create a secure, airtight connection to the filter plate. The plates have pins and holes for easy alignment and stability. 2

4 LIBRARY NORMALIZATION MAG-BIND EQUIPURE LIBRARY & gdna NORMALIZATION KITS Many high throughput applications, such as sequencing and genotyping, require the input DNA concentration to be within a certain range for optimal results. Traditionally, a tedious process of quantification, calculation and concentration adjustment must be carried out to normalize the DNA samples. The Mag-Bind EquiPure Normalization Kit completely eliminates the need to quantify and aliquot DNA, saving time, quantification bias and tip cost. Using our proprietary Mag-Bind Normalizer Beads and binding buffer system, input DNA of various quantities is simply bound, washed and eluted to a final normalized product. The magnetic beads have a limited binding capacity and therefore allow a pre-defined amount of DNA to be captured and eluted. An alternate protocol allows for non-bound DNA to be recovered. The Mag-Bind EquiPure Normalization Kit allows users to adjust the quantity of DNA that binds to the magneticc beads and elution volumes can be adjusted to vary DNA concentrations to user s needs. The Mag-Bind EquiPure Normalization Kit is fully automatable on multiple liquid handling platforms including Hamilton STAR, Tecan Evo, Caliper Sciclone and Beckman Coulter Biomek instruments. NORMALIZATION OF 16S NGS LIBRARIES NORMALIZATION OF RNA-SEQ NGS LIBRARIES DNA (ng) DNA (ng) Input DNA Samples Varying amounts of 16s NGS libraries were prepared and normalized with the Mag-Bind EquiPure Normalization Kit. DNA yield was quantified using Promega QuantiFluor dsdna system. Input DNA Samples Varying amounts of Illumina TruSeq Stranded mrna libraries were prepared and normalized with the Mag-Bind EquiPure Normalization Kit. DNA yield was quantified using Promega QuantiFluor dsdna system. 3

5 gdna NORMALIZATION NORMALIZED gdna qpcr FROM NORMALIZED gdna DNA Output (ng) , 2, ct , 2, DNA Input (ng) Genomic DNA was isolated from whole blood with the Mag-Bind Blood & Tissue DNA HDQ Kit. Varying amounts were inputted into the different reactions then normalized with Mag-Bind EquiPure gdna Kit. DNA yield was quantified using Promega QuantiFluor dsdna system. Mag-Bind EquiPure Library Normalization Kit Product Preparations M x 96 M x 96 Mag-Bind EquiPure gdna Normalization Kit Product Preparations M x 96 M x 96 4

6 DYE TERMINATOR REMOVAL MAG-BIND SEQDTR Omega Bio-tek s Mag-Bind SeqDTR is designed to effectively and reliably remove unincorporated terminators from sequencing reactions. Sequencing products are mixed with the Mag-Bind SeqDTR magnetic particles which selectively bind DNA. Two rapid wash steps eliminate trace contaminants such as nucleotides and primers to reduce background signal and therefore achieve higher QV scores. The high sensitivity of Mag-Bind SeqDTR s binding ability allows for decreased concentrations of BigDye chemistry to be used and longer continuous read lengths to be achieved. Mag-Bind SeqDTR can be processed in 96- and 384-well formats and is compatible with many liquid handling instruments including Hamilton STAR/STARlet, Beckman Coulter Biomek FX/NX, and Tecan Evo instruments. Up to four plates can be run in a 96-well format in less than 25 minutes. Mag Bind SEQ DTR Company C Company A 12 SIGNAL STRENGTH 9 CONTINUOUS READ LENGTH 6 QUALITY VALUE SCORE Comparison of magnetic bead-based big dye clean-up chemistries. Purified 1.8 kb PCR fragments were sequenced from each company using the recommended protocols. The median of 16 samples per company are used in the representations above. A 5 µl sequencing reaction was performed using a 1/32 dilution of Applied Biosystems Big Dye Terminator v3.1 Chemistry. DNA was analyzed on an Applied Biosystems 373XL. Mag-Bind SeqDTR Product Size M ml M ml M ml 5

7 PCR PURIFICATION MAG-BIND RXNPURE PLUS Omega Bio-tek s Mag-Bind RxnPure Plus Kit allows for the rapid and reliable recovery of DNA from PCR and other enzymatic reactions. The system combines Omega Bio-tek s proprietary chemistries with the reversible nucleic acid binding properties of magnetic beads to selectively bind PCR amplicons 1 bp and larger to eliminate excess nucleotides, primers and small, nontargeted amplification products, such as primer dimers. This kit is designed for manual and fully automated purification and may not require programming of current liquid handling instruments depending on your current method. Mag-Bind RxnPure Plus is compatible with multiple liquid handlers and magnetic processors such as Hamilton STAR /STARlet and Beckman Coulter Biomek FX/NX. RxnPure Plus Company A 5 BP Ladder Ten microliters of 5 bp ladder was purified with Omega Bio-tek s Mag-Bind RxnPure Plus Kit and a comparable kit from Company A, according to manufacturer s recommended protocols. The DNA was eluted in 2 microliters and analyzed on the Agilent Tapestation 22. Mag-Bind RxnPure Plus Product Size M ml M ml M ml 6

8 PLASMID DNA E-Z 96 FASTFILTER PLASMID DNA KIT The E-Z 96 FastFilter Plasmid DNA Kit combines the power of HiBind technology with the time-tested consistency of alkaline-sds lysis of bacterial cells to deliver high quality plasmid DNA. Using the E-Z 96 DNA plate, up to 96 samples can be processed in less than 9 minutes. The multi-layered E-Z 96 Lysate Clearance Plate is designed to eliminate time-consuming centrifugation steps for the clearance of bacterial alkaline lysates without clogging. With the combination of these two plates, this kit boasts an average DNA recovery rate of 1-3% higher than traditional manual centrifugation methods. The E-Z 96 FastFilter Plasmid DNA Kit typically yields 1-15 µg high-copy plasmid DNA from a 1 ml overnight culture, although yields will vary according to plasmid copy number, E. coli strain and growth conditions. The 96- well format of the E-Z 96 FastFilter Plasmid DNA Kit lends itself to automation on platforms equipped with vacuum manifolds such as Hamilton Nimbus, STAR and STARlet lines, Beckman Coulter Biomek FX and NX workstations and Tecan Freedom Evo systems. AUTOMATED EXTRACTION ON HAMILTON STAR MANUAL EXTRACTION E-Z 96 FastFilter Plasmid DNA Kit D197-1 x 96 D x 96 D x 96 Extraction Method Conc. (ng/µg) A 26 /A 28 A 26 /A 23 Automated Manual Comparison of automated and manual extraction methods using the E-Z 96 FastFilter Plasmid DNA Kit. Ninety-six 1.2 ml overnight cultures in 2xYT medium were lysed in a modified alkaline lysis system and cleared with an Omega Bio-tek E-Z 96 Lysate Clearance Plate. DNA was extracted from one half of the samples using the standard manual method and the other half using the Hamilton STAR liquid handling workstation. The samples were run on a 1% agarose gel. Concentration and quality was quantified using Thermo Fisher NanoDrop 2c. The table represents the average of 48 samples. 7

9 PLASMID DNA MAG-BIND ULTRA PURE PLASMID DNA 96 KIT The Mag-Bind Ultra Pure Plasmid DNA 96 Kit combines our alkaline lysis method with magnetic beads to offer a fast and economical option for high-quality plasmid DNA extraction. Two options are available to clear cellular debris after alkaline lysis: centrifugation or lysate clearing beads. The lysate clearing magnetic beads allow for fully automated processing after pelleting of the cells from culture. The kit utilizes Omega Bio-tek s endo-free plasmid DNA binding system for purification of high-quality DNA that is endotoxin-free. This flexible system can easily be adapted to most automated liquid handling platforms without sacrificing plasmid DNA quality. Plasmid DNA is suitable for restriction enzyme digestion, Sanger sequencing and transfection. ENDOTOXINS IN PLASMID DNA PREPS Prep Method EU/µg Mag-Bind Ultra-Pure Plasmid DNA Kit.5 Company P Magnetic Bead Plasmid DNA Kit Omega Bio-tek E.Z.N.A. Plasmid Mini Kit Endotoxins in plasmid DNA preps. Plasmid DNA was isolated from.8 ml LB culture from the corresponding method following the manufacturer s recommended protocols. Endotoxin level was determined with Thermo Scientific s Pierce LAL Chromogenic Endotoxin Quantitation Kit. PLASMID DNA YIELD COMPARISON Omega Bio-tek E.Z.N.A. Plasmid DNA Kit 1 Omega Bio-tek Mag-Bind Ultra Pure Plasmid DNA 96 Kit with lysate clearance via centrifugation Omega Bio-tek Mag-Bind Ultra Pure Plasmid DNA 96 Kit with lysate clearance via lysate clearing beads Company Q Silica Spin Columns Company P Magnetic Bead Plasmid DNA Kit Mag-Bind Plasmid DNA 96 Kit M x 96 M x 96 Plasmid DNA yield comparison. Plasmid DNA was isolated according to manufacturer s recommended protocols from.8 ml LB cultures grown for 24 hours. Each protocol was performed in triplicate with the average data shown above. DNA was quantified with Thermo Scientific NanoDrop 2c. 8

10 BLOOD, TISSUE, SALIVA & BUCCAL SWABS DNA MAG-BIND BLOOD & TISSUE DNA HDQ 96 KIT The Mag-Bind Blood and Tissue DNA HDQ 96 Kit is designed for the rapid and reliable isolation of highquality genomic DNA from 1-25 µl of blood, buccal swabs, buffy coat, or cultured cell samples. The magnetic response time of the Mag-Bind particles and the elimination of all heating steps allow for an overall rapid processing time. Using paramagnetic particles provides high-quality DNA that is suitable for direct use in most downstream applications such as qpcr, PCR, and next generation sequencing. The Mag-Bind Blood and Tissue DNA HDQ 96 Kit is compatible with multiple liquid handlers and magnetic processors such as Thermo KingFisher Flex, Qiagen BioSprint, Applied Biosystems/Ambion MagMAX 96, Beckman Coulter Biomek FX/NX, and Hamilton Star/Starlet. WHOLE BLOOD DNA ISOLATION FROM 1,5 µl qpcr FROM gdna ISOLATED FROM WHOLE BLOOD Sample Nucleic Acid Conc. A 26 /A 28 A 26 /A 23 Yield ct ng 5 ng.5 ng Template Amount Genomic DNA was extracted from 1,5 µl whole blood using Omega Bio-tek s Mag-Bind Blood and Tissue DNA HDQ 96 Kit. DNA was analyzed on a Thermo Scientific NanoDrop 2c. Fifty nanograms of DNA was diluted 1 and 1-fold and used as a template in a 2 microliter SYBR qpcr reaction. The ct values increased by only three cycles per 1-fold dilution, which demonstrates that the template DNA is free of inhibition. Mag-Bind Blood & Tissue DNA HDQ 96 Kit M x 96 M x 96 9

11 BLOOD, TISSUE, SALIVA & BUCCAL SWABS DNA Yield (µg) DNA YIELD FROM 2 µl WHOLE BLOOD Samples DNA yield from 2 µl whole blood. DNA yield from 2 µl whole blood extracted using the Mag-Bind DNA HDQ Kit on the ABI MagMAX 96 instrument. DNA yield was determined by PicoGreen quantification. SALIVA DNA EXTRACTION Kit Biomatrica Oragene Omega Bio-tek Company A Saliva DNA extraction. DNA yield from 2 µl saliva samples stabilized in Biomatrica s DNAguard and DNA Genotek Oragene saliva tubes. DNA was extracted in quadriplicate according to the manufacturer s recommended protocols and eluted in equal amounts. DNA yield was determined by PicoGreen quantification and average DNA concentration is shown. BUFFY COAT DNA EXTRACTION Sample Yield UV Yield flourescent dye Omega Bio-tek Omega Bio-tek Omega Bio-tek Company T Company T Company T BUCCAL SWABS DNA EXTRACTION COMPARISON Omega Bio-tek Company A Buffy coat DNA extraction. 1, µl buffy coat samples DNA was extracted in triplicate according to the manufacturer s recommended protocols and eluted in equal amounts. DNA yield was determined by Promega Quantus dsdna quantification kit (flourescent dye) and Thermo Scientific Nanodrop 2c (UV). Genomic DNA was extracted from buccal swab samples using Omega s Mag-Bind Blood DNA HDQ 96 Kit and Company A s genomic DNA extraction kit. Ten percent of DNA extracted from each sample was analyzed on a.8% agarose gel. 1

12 VIRAL DNA & RNA MAG-BIND VIRAL DNA/RNA 96 KIT The Mag-Bind Viral DNA/RNA 96 Kit isolates DNA and RNA from serum, plasma, swabs, cell culture supernatants, urine and other acellular body fluids. This kit uses a unique system to efficiently lyse viral samples. The high binding ability and sensitivity of the magnetic particle technology allows efficient isolation from low titers or large sample sizes. The samples are lysed in a specially formulated buffer containing detergent. Binding conditions are adjusted to enable the nucleic acid to bind to the Mag-Bind particles. After proteins and cellular debris are efficiently washed away, pure RNA and DNA is eluted in nuclease-free water or Elution Buffer. This system can be used on multiple automated platforms including Ambion MagMAX, Thermo KingFisher Flex, Beckman Coulter Biomek FX, and Hamilton STAR instruments. ct Omega Bio-tek Company A HBV virus (in quantities of 1 and 1 infectious unit(s)) was spiked into 2 µl of human serum. Viral nucleic acid was isolated with Omega Bio-tek s Mag-Bind kit and with a comparable kit from Company A according to recommended protocols. 5 µl of template was used for a SYBR Green labeled qpcr reaction, which was replicated four times. The resulting mean ct values are shown in the above figure. Mag-Bind Viral RNA/DNA 96 Kit M x 96 M x 96 M x 96 RNA Template 1 x 1 7 viral particles/µl 1 x 1 6 viral particles/µl 1 x 1 5 viral particles/µl 1 x 1 4 viral particles/µl 1 x 1 3 viral particles/µl ct Value ct values of recovered RNA isolated using the Mag-Bind Viral RNA/DNA 96 Kit. Serum was separated from a human blood sample containing 1 x 1 7 Hepatitis B viral particles/ µl. A 1-fold dilution series of the serum was performed and 5 µl of each dilution was used in the Mag-Bind Viral RNA/ DNA 96 Kit to isolate viral RNA. Two µl RNA was used as the template. 11

13 PATHOGEN DNA & RNA MAG-BIND UNIVERSAL PATHOGEN KIT The Mag-Bind Universal Pathogen 96 Kit allows for the rapid and reliable isolation of high quality host genomic DNA, Gram-positive and negative bacterial DNA, fungal spore DNA, viral DNA and viral RNA from tissue, blood, urine, serum and fecal samples. The system allows for automation after sample lysis via Hamilton STAR, Thermo KingFisher Flex, Applied Biosystems MagMAX, Qiagen BioSprint and other liquid handling instruments. E-Z 96 Disruptor Plus plates are included with the kit for simultaneous homogenization and lysis of the sample. The unique buffer system does not require alcohol to bind nucleic acids, allowing recovery of high quality DNA/RNA free of PCR inhibitors. The entire procedure can be completed in less than 6 minutes for 96 samples. 3 FUNGAL SPORES Undiluted 1:1 1:1 Average ct GRAM-POSITIVE BACTERIA Stool Urine Undiluted 1:1 Group B strep cultured samples were added to corresponding sample types and isolated with Mag- Bind Universal Pathogen Kit. 2 µl SYBR qpcr were performed in triplicate on primers specific to the target organism. Average of triplicate data shown. Average ct VIRAL RNA Undiluted 1: Serum + Crypto Stool + Crypto Urine + Crypto Cryptosporidium oocysts were added to corresponding sample types and isolated with Mag-Bind Universal Pathogen Kit. 2 µl SYBR qpcr were performed in triplicate on primers specific for the target organism. Average of triplicate data shown. Average ct Mag-Bind Universal Pathogen 96 Kit M429-1 x 96 M x 96 Stool Urine Serum HBV viruses were added to corresponding sample types and isolated with Mag-Bind Universal Pathogen Kit. 2 µl SYBR qpcr were performed in triplicate on primers specific for the target organism. Average of triplicate data shown. 12

14 FFPE DNA FFPE DNA MAG-BIND FFPE DNA 96 KIT The Mag-Bind FFPE DNA 96 Kit is designed for the rapid and reliable isolation of DNA from formalinfixed, paraffin-embedded (FFPE) tissue. The specially formulated buffers reverse crosslinking and release short and long DNA fragments. Mag-Bind particles allow for automation-friendly extraction from FFPE tissue in under 6 hours. Purified DNA is suitable for downstream applications including SNP analysis, sequencing and genotyping. This system is fully compatible on Hamilton STAR, Beckman Coulter Biomek FX, Biomek 2, Tecan Fredom Evo, Thermo KingFisher Flex and other workstations. ct Value qpcr FROM FFPE DNA Omega Bio-tek Mag Bind FFPE DNA Company A Spin Column 25 Company A Samples Yield (µg/ section) A 26 / 28 A 26 / Averages Averages DNA (ng) ct values of recovered DNA using the Mag- Bind FFPE DNA Kit and Company A Spin Column Kit for FFPE DNA purification. A 1-fold dilution series of the recovered DNA was used in a SYBR Green-based real-time PCR reaction targeting an 8-bp fragment of the human ribosomal protein gene RPL13a. Each reaction was performed in triplicate. The input DNA amounts were 5, 5,.5, and.5 ng, respectively. Comparison of Company A s silica spin column method for FFPE DNA purification versus Omega Biotek s Mag-Bind FFPE DNA Kit. Both protocols were performed according to manufacturer s recommened protocols. Samples were quantitated using Thermo Fisher NanoDrop 2c. Mag-Bind FFPE DNA 96 Kit M x 96 M x 96 13

15 FFPE RNA MAG-BIND FFPE RNA 96 KIT The Mag-Bind FFPE RNA 96 Kit is designed to isolate total RNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. This kit uses Mag-Bind technology for the efficient isolation of total RNA along with optimized buffers that are designed to minimize the effects of formaldehyde modifications. Two options are available for paraffin removal: a heat treatment or a xylene treatment. Samples can be processed manually or on the Hamilton STAR and STARlet, Beckman Coulter Biomek FX, Biomek 2, Tecan Freedom Evo, Velocity Biocell and Thermo KingFisher Flex workstations. MAG-BIND FFPE RNA 96 KIT PROTOCOL Lyse sample and remove paraffin Prepare lysate Add Mag-Bind Particles for RNA binding and magnetize. Aspirate supernatant containing contaminants. Wash Mag-Bind Particles Sample Type Human Breast Tissue Human Kidney Rat Liver Source RNA Yield (µg) Increase Number Company A Omega Bio-tek % % % % % % % % % % RNA yield from various sample types using Mag- Bind FFPE RNA Kit versus Company A on Thermo KingFisher Flex. Both protocols were performed according to manufacturer s recommended protocols. The Omega Bio-tek system provides better sensitivity for gene expression profiling. % Magnetize Add DNase, wash, and magnetize Dry Mag-Bind Particles on magnet Mag-Bind FFPE RNA 96 Kit M x 96 M x 96 Elute RNA 14

16 BLOOD DNA & RNA E-Z 96 BLOOD & TISSUE DNA KIT Omega Bio-tek s E-Z 96 Blood & Tissue DNA Kit provides an easy and rapid method for the isolation of genomic, mitochondrial or viral DNA. Up to 25 µl fresh, frozen or anti-coagulated whole blood can be readily processed at one time from common anticoagulants such as EDTA, heparin and sodium citrate. The E-Z 96 Blood & Tissue DNA Kit can also be used for the preparation of genomic DNA from buffy coat, serum, plasma, saliva, cultured cells, buccal swabs and other bodily fluids. An optimized lysis buffer and protease digestion step removes nucleases, histones and other proteins. After binding conditions are adjusted and DNA is bound to the HiBind matrix, the DNA is washed and PCR inhibitors are removed. High quality DNA is eluted and can be used for downstream applications such as PCR, qpcr and microarray analysis., A 26 /A 28 DNA yield (μg) QUALITY & YIELD OF GENOMIC DNA PURIFIED Samples E-Z 96 Blood DNA Kit D x 96 D x Samples Quality and yield of genomic DNA purified using the E-Z 96 Blood & Tissue DNA Kit. Purified genomic DNA from 2 µl whole human blood was isolated with the E-Z 96 Blood & Tissue DNA Kit. DNA was eluted in 1 µl and quantitated using Thermo Fisher NanoDrop 2c. MAG-BIND PX BLOOD RNA 96 KIT The Mag-Bind PX Blood RNA 96 Kit provides a convenient and fast RNA extraction solution for samples stabilized with PAXgene Blood RNA tubes or Applied Biosystems Tempus Blood RNA tubes. This system combines Mag-Bind particles and HiBind silica technologies for purification of total RNA from up to 2.5 ml preserved blood samples. The blood sample is spun down and the crude RNA/DNA pellet is collected and washed. The pellet is then resuspended and digested with proteinase K. The lysate is applied to a filter plate to remove genomic DNA. This procedure completely removes inhibitors and sample stabilization reagents to allow for reliable downstream analysis. High-quality purified RNA can be used for downstream applications such as qrt-pcr, RT-PCR, and microarray analysis. 15 Mag-Bind PX Blood RNA 96 Kit M x 96 M x 96

17 TOTAL RNA E-Z 96 TOTAL RNA KIT The E-Z 96 Total RNA Kit is designed for the isolation of total cellular RNA from up to 5 x 1 6 cultured cells or soft tissues. This kit can process single or multiple samples in less than 6 minutes. Samples are lysed in a denaturing lysis buffer which inactivates RNases. Binding conditions are adjusted and then lysate is transferred to a 96-well HiBind RNA plate where the RNA is purified via three wash steps. High-quality RNA is eluted in RNase-free water. RNA purified using the E-Z 96 Total RNA method is ready for applications such as RT-PCR, qpcr, differential display, microarrays, and other downstream applications. E-Z 96 Total RNA Kit R134-1 x 96 R x 96 R x 96 RNA Yield (µg) RNA YIELD FROM TISSUE Liver Kidney Spleen Heart Muscle Lung Yield of isolated RNA with the E-Z 96 Total RNA Kit. Purified total RNA from 2 mg various animal tissues was isolated with the E-Z 96 Total RNA Kit using a Thermo KingFisher Flex. RNA was quantitated using a Thermo Fisher NanoDrop 2c. MAG-BIND TOTAL RNA 96 KIT The Mag-Bind Total RNA 96 Kit is designed for the rapid and reliable isolation of total cellular RNA from cultured cells and soft tissues. The system utilizes the high nucleic acid binding properties of Mag-Bind particles resulting in increased sensitivity over silica column-based systems. Mag-Bind particles are completely resuspended during the wash steps to increase contact with wash buffers and enhance removal of contaminants resulting in high-quality RNA. RNA ISOLATION FROM SMALL SAMPLE AMOUNTS Yield (µg) Mag-Bind Total RNA 96 Kit M x 96 M x 96 5K 1K 5K 1K RNA Isolation from small sample amounts. Total RNA from corresponding cell number amount HEK293 cultured cells using the Mag-Bind Total RNA 96 Kit. RNA was quantified using Promega QuantiFluor. 16

18 PLANT DNA E-Z 96 PLANT DNA DS KIT The E-Z 96 Plant DNA DS Kit is designed for the efficient recovery of genomic DNA up to 3 kb in size from fresh, frozen or dried plant tissue samples rich in polysaccharides, polyphenols, or having a lower DNA content. Up to 5 mg of wet tissue can be processed in less than 1 hour. The system combines the reversible nuclec acid binding properties of the HiBind matrix with the speed and versatility of spin column technology to eliminate polysaccharides, phenolic compounds, and enzyme inhibitors from plant tissue lysates. Purified DNA is suitable for PCR, restriction enzyme digestion and hybridization applications. This procedure relies on the well-established properties of the cationic detergent, cetyltrimethyl ammoniumbromide (CTAB), in conjunction with the unique binding system to increase yields and provide high-quality DNA. The system eliminates the need for chloroform extractions traditionally associated with CTAB-based lysis methods. Samples are homogenized and lysed in a high salt buffer containing CTAB and binding conditions are adjusted and DNA is purified using a HiBind DNA matrix. Salts, proteins, anad other contaminants are removed to yield high-quality genomic DNA suitable for downstream applications such as endonuclease digestion, thermal cycle amplification and hybridization applications COMPARISON OF DNA YIELDS FROM MULTIPLE CROPS Omega Bio-tek Company Q E-Z 96 Plant DNA DS Kit D x 96 D x 96 ng/mg Canola Corn Cotton Soybean Tobacco 4-5 mg of respective fresh leaf tissue were extracted in triplicate according to the manufacturer s recommended protocol and eluted in 1 µl. DNA analyzed with fluorescent DNAbased quantfication method. Total yield was divided by total tissue amount to show ng of DNA per mg of leaf tissue. 17

19 PLANT MAG-BIND PLANT DNA DS 96 KIT The Mag-Bind Plant DNA DS 96 Kit is designed for difficult samples which are hard to lyse or contain high amounts of polysaccharides and polyphenols. This system uses a CTAB-based lysis buffer which does not require organic solvents. The proprietary binding system prevents inhibitors from binding to the magnetic beads. The wash buffer system further removes polysaccharides, phenolic compounds, and enzyme inhibitors from plant tissue lysate. An optional rebinding step is included with the kit, but most samples do not require the additional treatment. Using paramagnetic particles provides highquality DNA that is suitable for direct use in most downstream applications such as qpcr, PCR and next generation sequencing. The Mag-Bind Plant DNA DS 96 Kit is compatible with multiple liquid handlers and magnetic processors such as Thermo KingFisher Flex, Qiagen BioSprint, Applied Biosystems/Ambion MagMAX 96, Beckman Coulter Biomek FX/NX, and Hamilton STAR/STARlet. DNA YIELD COMPARISON Type Omega Bio-tek Company Q Canola Corn Cotton Potato Soybean Tobacco Wheat Approximately 5 mg leaf sample extracted per sample according to manufacturer s recommended protocols. DNA concentration determined via flourescence-based nucleic acid quantification. DNA quantification confirmed via SYBR qpcr (data not shown). Amount of DNA per mg leaf sample shown above. Mag-Bind Plant DNA DS 96 Kit M113-1 x 96 M x 96 E-Z 96 PLANT RNA KIT The E-Z 96 Plant RNA Kit provides a convenient and rapid method for the isolation of total RNA from a variety of plant samples. This kit provides an E-Z 96 Homogenizer Plate for the homogenization and filtration of viscous plant cell lysates in combination with the HiBind RNA Plate for RNA purification. The unique lysis buffer formulation contains chaotropic salts and detergents for efficient removal of contaminants including polysaccharides and phenolic compounds. Purified RNA can be used for most downstream applications such as RT-PCR, qpcr, Northern blot analysis, differential display, microarray analysis, whole transcriptome sequencing and poly A+ RNA selection. E-Z 96 Plant RNA Kit R x 96 R x 96 Samples validated with the E-Z 96 Plant RNA Kit Arabidopsis thaliana Beta vulgaris Daucus carota Euglena gracilis Fragaria virginiana Hordeum vulgare Humulus lupulus Lycopersicon esculentum Nicotiana tabacum Ornithogalum thyrsoides Oryza sativa Solanum tuberosum Spinacia oleracea Triticum aestivum Zea mays 18

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