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Nucleic Acid Purification
DNA Purification. Genomic
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MasterPureTM

Genomic DNA Purification Kit

MasterPure™ Complete DNA and RNA Purification Kit

Applications
  • Purification of TNA, DNA, or RNA for many applications, including PCR, RT-PCR, cloning, sequencing, Northern and Southern blotting, restriction analysis, and genomic library preparation.

The MasterPure™ Complete DNA and RNA Purification Kit uses a novel technology that enables efficient purification of intact total nucleic acid (TNA), DNA, or RNA from every type of biological material (Table 1 and Fig. 1).

The gentle salt-precipitation protocol permits rapid purification of high-molecular-weight nucleic acids from a single sample or many samples that are processed simultaneously (Fig. 4). High yields of nucleic acids are obtained using MasterPure Kits (Table 2), and yields and purity are highly consistent for different samples of the same material (Fig. 2). The resultant nucleic acids have an A260/A280 ratio of 1.8-2.0, indicating that they are substantially free of protein. The TNA, DNA, and/or RNA purified from different samples can be used directly in many applications. For example, the sensitivity of PCR or RT-PCR assays is often improved if the template is obtained using a MasterPure Purification Kit.

Benefits
  • Purify TNA in 30 minutes without cumbersome columns.
  • A260/A280 ratios consistently between 1.8 and 2.0.
  • Recover >90% of theoretical DNA yield.
  • No phenol, chloroform, or other caustic solvents.
  • Each kit component is also available separately in a 10X package size.
  • Low- and high-molecular-weight nucleic acids are recovered.
Reference
  1. Schanke, J. and Watson, J. (1998) Epicentre Forum 5(2), 12.

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Figure 1. Overview of the MasterPure™ Complete Kit protocol.

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Figure 2. Comparison of PCR sensitivity using templates obtained using the MasterPure™ Complete DNA and RNA Purification Kit versus a spin-column kit. PCR amplification was performed after extraction from serial dilutions of E. colicells ranging from 2 x 107 (lanes 1) to 200 (lanes 6). Lane M, DNA marker; lane 7, negative control.
 g_masterpure_completekit_6gels.jpg
Figure 3 (click to enlarge). DNA, RNA, and total nucleic acid (TNA) purified from diverse cell sources using the MasterPure™ Complete Kit. M = kb ladder.Table 1. Examples of targets and samples analyzed using MasterPure™ DNA and RNA Purification Kits. Using an extremely simple salt-precipitation protocol, TNA, DNA, or RNA can be isolated from virtually any source in less than 1 hour.
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*For extraction of yeast DNA, the MasterPure™ Yeast DNA Purification Kit is recommended
 

Table 2. Example of yields of nucleic acids obtained using MasterPure™ Complete DNA and RNA Purification Kits.











 Table 1. Examples of targets and samples analyzed using MasterPure™ DNA and RNA Purification Kits. Using an extremely simple salt-precipitation protocol, TNA, DNA, or RNA can be isolated from virtually any source in less than 1 hour.
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 Figure 4. Consistent purification of DNA, TNA, and RNA.Five different genomic DNA (lanes 1-5), TNA (lanes 6-10), and total cellular RNA (lanes 11-15) samples were isolated from frozen bovine liver with the MasterPure™ Complete DNA and RNA Purification Kit. Lane M, Kilobase ladder.  



MasterPure™ Plant Leaf DNA Purification Kit

Applications
  • Purification of plant-leaf DNA for microsatellite typing, PCR amplification, restriction digestion, Southern blotting, and other molecular biology applications.

The MasterPure™ Plant Leaf DNA Purification Kit is designed for purifying DNA from 35-100 mg of fresh plant-leaf tissue. The protocol can be completed in less than 1 hour and may be scaled up to obtain larger amounts of DNA. The purified DNA can be used for PCR amplification, restriction digestion, Southern blotting, or other molecular biology applications.

The MasterPure Kit was designed for even the most difficult plant leaf DNA purifications. The kit was optimized with the grapevine, Vitis vinifera, whose leaves are notoriously high in polyphenolic (tannin-like) compounds and polysaccharides, which can interfere with both the isolation and subsequent use of DNA. Plants tested include grape, apple, tomato, maize, sugarcane, sunflower, fern, quillwort, and pine.

Benefits
  • Recover DNA from the leaves of many plant species, even those high in polysaccharides and polyphenolic compounds.
  • Purify DNA in less than 1 hour.
  • Yields high-molecular-weight DNA (Fig. 1).
  • No organic solvent or CTAB extractions, CsCl gradients, or enzyme digestions.
  • Recover DNA of higher integrity than column-based purification methods (Fig. 2).

Product Citations
  1. Bowers, J.E. et al. (1993) Am. J. Enology and Viticulture 44, 266.
  2. John, M.E. (1992) Nucl. Acids Res. 20, 2381.
  3. Thomas, M.R. et al. (1994) Plant Mol. Biol. 25, 939.
  4. Rowland, L.J. and Nguyen, B. (1993) BioTechniques 14, 735.
  5. Bowers, J.E. et al. (1996) Genome 39, 6328.
  6. Hoffman, L. and Moan, E. (1999) Epicentre Forum 6(1), 1.

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 Figure 1. Comparative yields of DNA isolated with the MasterPure™ Kit and another supplier's kit. A 10-µl aliquot of each DNA preparation (either MasterPure DNA or Supplier G) were separated by electrophoresis in a 0.7% agarose gel; the gel was stained with SYBR® Gold and viewed with a Dark Reader™ Transilluminator. Lanes M, 1-kb DNA ladder; lanes 1-4, Pinot noir grape leaf DNA purified with the MasterPure Kit; lanes 5-8, Pinot noir grape leaf DNA purified with Supplier G Kit. HMW DNA, high-molecular-weight DNA. Figure 2. Restriction digests of DNA isolated with the MasterPure™ Kit versus a column-based kit. Lane 2 shows tobacco leaf DNA purified with the MasterPure Plant Leaf DNA Purification Kit, digested with Ssp I. Lane 3 shows the same digestion using DNA purified with Supplier Q's column-based kit. DNA purified with the MasterPure Kit was cleaved by Ssp I, as seen by the existence of a repetitive element band. The repetitive element is not cleaved by Ssp I using DNA purified with the Supplier Q Kit. Lanes 1 and 4, DNA markers. 



MasterPure™ Yeast DNA Purification Kit

Poster on rapid fungal DNA typing from ASM 2006 (2.3 MB)

Applications
  • Purification of DNA from yeast for many molecular biology applications, including PCR amplification, restriction endonuclease digestion, Southern blotting, and genomic library preparation.
  • Identification and typing of fungi.

The MasterPure™ Yeast DNA Purification Kit enables efficient, high-yield purification of high-molecular-weight DNA from yeast and other fungi. The protocol involves nonenzymatic cell lysis at 65°C, followed by removal of protein by precipitation, and nucleic acid precipitation and resuspension. No lyticase, proteolytic enzymes, or bead-beating are used in the procedure. Yeast genomic DNA yields using the MasterPure Kit are much higher than yields obtained with other commercially available kits (Fig. 1).1 The protocol can be easily adjusted for larger or smaller samples, including single yeast colonies.1 The recovered nucleic acid can be used directly in most applications, including PCR amplification.

Benefits
  • Higher yields of yeast chromosomal DNA than with other kits.
  • Recover DNA from a wide variety of yeast species, includingCandida, Saccharomyces, Pichia, and Schizosaccharomyces, and filamentous fungi such as Aspergillus
  • 2 and Penicillium.
  • Purify DNA in less than 40 minutes.
  • No enzymatic lysis, columns, or phenol or other organic extractions. No bead-beating.
  • The recovered DNA has a high molecular weight (Fig. 2), and is suitable for many applications, including PCR amplification.

References
  1. Hoffman, L. and Moan, E. (1998) Epicentre Forum 5(4), 1.
  2. Jin, J., Lee, Y.-K. and Wickes, B.L. (2004) J. Clin Microbiol. 42, 4293.
g_masterpureyeast_sizedistdna.jpg 
 Figure 2. Size distribution of DNA produced from the MasterPure™ Kit.For each kit indicated, a 500-ng aliquot of purified yeast DNA was analyzed by pulse-field gel electrophoresis on a 1% agarose gel. The gel was stained with ethidium bromide. The majority of DNA isolated with the MasterPure Kit was estimated to be in the 40- to 50-kb size range, while degradation to smaller fragments was observed with other kits. Lane M, lambda DNA ladder; Lane 4, T7 DNA (40 kb).
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 Figure 1. Comparative yields of DNA from the MasterPure™ Yeast DNA Purification Kit and competitor's kits. DNA was quantitated specifically with Hoechst fluorescent dye 33258, which gives minimal fluorescence with RNA. The data represent the average DNA yields determined by fluorometry from two experiments with S. cerevisiae and C. albicans. The MasterPure Kit produced up to 17 times more DNA from C. albicans and 12 times more DNA from S. cerevisiaethan other kits.


MasterPure™ Gram Positive DNA Purification Kit

Applications
  • Purification of genomic DNA from Gram-positive bacteria for fosmid library construction, PCR, restriction digests, and Southern blotting.

The MasterPure™ Gram-Positive DNA Purification Kit provides all of the reagents needed to purify DNA from Gram-positive bacteria. These bacteria lyse more readily after treatment with Ready-Lyse™ Lysozyme and the Gram-Positive Cell Lysis Solution included in the kit. Ready-Lyse Lysozyme is a stable solution of a recombinant lysozyme from a nonmammalian, nonavian source. It has high specific activity and does not bind DNA.1,2
Examples of Gram-positive bacteria tested with this kit are listed in Table 1. The size range of DNA purified by the MasterPure Gram-Positive DNA Purification Kit is shown in Fig. 1. The DNA purified using the kit is suitable for PCR analysis (Fig. 2), and other molecular biology applications.

Benefits
  • PCR-ready DNA.
  • Scaleable method for large sample volumes.
  • High-molecular-weight DNA
  • Lysozyme included; no separate purchase required.
  • Tested on a variety of species.
  • Gram-negative bacteria are also lysed.

References
  1. Hoffman, L. and Jarvis, B. (2003) Epicentre Forum 10(3), 3.
  2. Jarvis, B. and Hoffman, L. (2004) Epicentre Forum 11(3), 7.
  3. Luna, V.A. et al. (2006) J. Clin. Microbiol. 44, 2367.
  4. Li, J. et al. (2007) Immunol. 75, 1811.
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 Table 1. Incubation times needed for DNA recovery from 1 ml of Gram-positive bacterial culture.

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 Figure 1. Electrophoretic analysis of DNA purified using the MasterPure™ Gram-Positive DNA Purification Kit. Panel A: DNA purified from B. subtilis (ATCC 6051) was separated on a 1% agarose gel and stained with SYBR® Gold. Lane M, kilobase ladder; lane 1, 300 ng of B. subtilis DNA. Panel B: Pulse-field gel electrophoresis of B. subtilis DNA. Lane M, Phage lambda ladder; lane 1, 300 ng of DNA; lane 2, 600 ng of DNA.








g_masterpuregram_pcranalysis.jpg
 Figure 2. PCR analysis of DNA purified using the MasterPure™ Gram-Positive DNA Purification Kit. B. subtilis DNA was amplified by PCR using BacF/ BacR primers, and the products were analyzed by agarose gel electrophoresis. Lane M, 100-bp ladder; lane 1, PCR product from 0.5 ng of B. subtilis DNA; lane 2, no-template control.





Meta-G-Nome™ DNA Isolation Kit

Applications
  • Isolation of fosmid cloning-ready metagenomic DNA from microbes in water, soil, and compost samples.
  • Isolation of metagenomic DNA for PCR or next-generation sequencing.

Note: The Meta-G-Nome™ DNA Isolation Kit replaces Epicentre's Metagenomic DNA Isolation Kit for Water.
The Meta-G-Nome™ DNA Isolation Kit is used to isolate inhibitor-free, fosmid cloning-ready DNA from unculturable or difficult-to-culture microbial species present in environmental water, soil, or compost samples (Fig. 1). The kit isolates high-molecular-weight DNA that is randomly sheared (with fragment sizes approximately 40 kb), does not require size selection, and can be used directly for end-repair and construction of fosmid libraries, PCR amplification, or next-generation sequencing. The kit can also be used to prepare 16S rRNA metagenomic libraries for phylogenetic classification or species-abundance studies.

The procedure uses filtration technology (with 1.2-µm and 0.45-µm membranes provided) and enzymatic lysis (with Ready-Lyse™ Lysozyme and Proteinase K) to isolate metagenomic DNA from water (100 ml per extraction), soil (1 g per extraction), or compost (300 mg per extraction). The purified DNA is free from humic and fulvic acids.

Benefits
  • No bead-beating, agarose plugs, pulsed-field gel electrophoresis, or size selection.
  • No phenol/chloroform extractions or CTAB.

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Figure 1. Schematic overview of the procedure for isolating DNA from soil or water samples using the Meta-G-Nome™ DNA Isolation Kit.

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 Figure 2. Size of inserts from a metagenomic fosmid library. Meta-genomic DNA isolated from lake water was used to generate a fosmid library using the CopyControl™ Fosmid Library Production Kit. DNA was isolated from randomly selected clones and digested with Not I. The insert sizes were approximately 40 kb. Lane M, Kilobase ladder; lane 1, 40-kb control insert; lanes 2-19, Not I-digested fosmid DNA.  Figure 3. PCR amplification of soil metagenomic DNA.DNA was isolated following the kit protocol, and PCR was performed using 16S rRNA primers using various dilutions of the template DNA. Lane M, Kilobase ladder; lanes 1 and 2, 1 µl and 5 µl of PCR products from 1:10 template dilution; lanes 3 and 4, 1 µl and 5 µl of PCR products from 1:100 template dilution; lane 5, 1 µl of PCR products from undiluted template; lane 6, 1:10 dilution of sample from lane 5.



Metagenomic DNA Isolation Kit for Water


Applications
  • Easy isolation of fosmid cloning-ready metagenomic DNA from microbes, including Gram-positive bacteria, in a water sample.
  • Isolation of metagenomic DNA for PCR or next-gen sequencing.

The Metagenomic DNA Isolation Kit for Water was developed to isolate metagenomic DNA directly from culturable or unculturable microbes in a water sample, to generate cloning-ready DNA for construction of random-sheared fosmid libraries. Traditional methods for isolation of metagenomic DNA to be used in fosmid library construction involve agarose plugs, random shearing, and a size selection using pulse-field gel electrophoresis before cloning into a fosmid vector. These methods (Fig. 1B) are time-consuming, inefficient, and result in a library with biased genome representation. The Metagenomic DNA Isolation Kit for Water isolates high-molecular-weight DNA that is randomly sheared (with fragment sizes approximately 40 kb), does not require size selection (Fig. 1A), and can be used directly for end-repair and cloning into the Epicentre pCC1FOS™ Vector (CopyControl™ Fosmid Library Production Kit, sold separately).

The kit protocol uses Ready-Lyse™ Lysozyme followed by Proteinase K treatment to isolate high-molecular-weight DNA. Using this kit, DNA isolated from 300 ml of lake water can result in a fosmid library containing as many as 100,000 metagenomic clones.

Benefits
  • No bead-beating.
  • No agarose plugs, mechanical shearing, or size selection.
  • Prevents loss of DNA, thereby generating unbiased libraries.
  • No columns or phenol/chloroform extractions.
  • Fosmid cloning-ready DNA (Fig. 2) in 90 minutes.
  • Improved yield, increasing the probability of recovering DNA from low-abundant organisms present in the collective genomes in environmental samples.

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Figure 1 . Comparison of traditional and Epicentre procedures for metagenomic DNA isolation and library construction.

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 Figure 2. Size of inserts from a metagenomic fosmid library.Metagenomic DNA isolated from lake water was used to generate a fosmid library using the CopyControl™ Fosmid Library Production Kit. DNA was isolated from randomly selected clones and digested with Not I. The insert sizes were approximately 40 kb. Lane M, Kilobase ladder; lane 1, 40-kb control insert; lanes 2-19, Not I-digested fosmid DNA.
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