Microbial Genomics

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In Vivo Transposomics™ Research

EZ-Tn5™ <KAN-2>Tnp and EZ-Tn5™ <DHFR-1>Tnp Transposome™ KitsRapidly generate knock-in or knock-out mutations

EZ-Tn5 Transposome complexes are formed between an EZ-Tn5 Transposon and EZ-Tn5 Transposase, and provide a simple and reliable method for generating a library of random gene knockouts in vivo.* The kits can also be used for knock-in of genes for bacterial strain development, tagging bacteria for environmental localization studies, and direct sequencing of bacterial chromosomal DNA.

Just electroporate the EZ-Tn5 Transposome into any of a broad range of living bacterial cells and select for a marker encoded by the EZ-Tn5 Transposon (Fig. 1).

• Rapid mutagenesis procedure is simpler and easier to use than chemical mutagenesis.• More efficient than using mini-transposons with suicide plasmids.• Broad host range: over 60 species of Gram-negative and Gram-positive bacteria

transposed so far.

*The use of Transposome™ complexes for in vivo insertion of a transposon, including, but not limited, to HyperMu™ and EZ-Tn5™ Transposome™ complexes, is covered by U.S. Patent No. 6,159,736 and related patents and patent applications, exclusively licensed to Epicentre. See www.epicentre.com/legal.

EZ-Tn5™Transposome

Cells

Sequencing

Insertion Clones1. Electroporate

2. PlateSelection Plate

Genomic DNA

Figure1.Overviewofin vivotransposition.

The EZ-Tn5™ Transposon insertion site in bacterial DNA can be sequenced directly using genomic DNA isolated using the MasterPure™ Complete DNA Purification Kit and primers homologous to the ends of the transposon.

Cat.# Quantity

EZ-Tn5™ <KAN-2>Tnp Transposome™ KitTSM99K2 10 Reactions

EZ-Tn5™ <DHFR-1>Tnp Transposome™ KitTSM99D1 10 ReactionsContents: Ready-to-use Transposome complex with either a KAN-2 or DHFR-1 marker, and forward and reverse primers for sequencing.

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In Vivo Transposomics™ Research

Cat.# Quantity

EZ-Tn5™ <R6Kγori/KAN-2>Tnp Transposome™ KitTSM08KR 10 ReactionsContents: EZ-Tn5 <R6Kγori/KAN-2>Tnp Transposome, KAN-2 FP-1 Forward Primer, R6KAN-2 RP-1 Reverse Primer, Sterile Water.

Figure1.Overviewofrescuecloningoftransposoninsertionsites.

<R6Kγori/KAN-2> clone Purify, then shear or digest genomic DNA

Self-ligation

RKan

ori

Rescued plasmid DNA KanR rescued clones

Transform pir E. coli and select on Kan plates

RKanori

RKan

ori

RKanori

RKanori

RKanori

RKanori

EZ-Tn5™ <R6Kγori/KAN-2>Tnp Transposome™ KitSimplify rescue cloning of genomic DNA or plasmids

Nothing makes the cloning process easier than creating mutations in vivo with the EZ-Tn5 <R6Kγori/KAN-2>Tnp Transposome Kit.* In addition to encoding a broad host-range kanamycin-resistance gene, the transposon contains an E. coli conditional origin of replication (R6Kγori). The presence of this origin of replication enables the propagation or “rescue” of the region of genomic DNA, or plasmid, into which the transposon has been inserted (Fig. 1).

• Easily recover and propagate plasmids from diverse bacterial genera that will not normally replicate in E. coli.

• Simple rescue cloning process of mutagenized genes speeds up structure/function studies and sequencing.

• Random insertion of transposon DNA assures excellent coverage of the entire bacterial chromosome.

• Rescue clones can be sequenced bidirectionally using the primers provided that are homologous to the ends of the transposon.

*The use of Transposome™ complexes for in vivo insertion of a transposon, including, but not limited, to HyperMu™ and EZ-Tn5™ Transposome™ complexes, is covered by U.S. Patent No. 6,159,736 and related patents and patent applications, exclusively licensed to Epicentre. See www.epicentre.com/legal.

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In Vivo Transposomics™ Research

EZ-Tn5™ Custom Transposome™ Construction KitsCreate your own EZ-Tn5 Transposomes using almost any DNA

The new EZ-Tn5 Custom Transposome Construction Kits provide the key reagents needed for constructing your own Transposomes. Also included are the standard pMOD PCR primers, 10X EZ-Tn5 Reaction Buffer for in vitro reactions, detailed protocols for creating and purifying the custom transposons, and stop solution for in vitro reactions. Sufficient reagents are provided to create Transposomes for up to 10 in vitro or 20 in vivo transposition reactions.

Cat.# Quantity

EZ-Tn5™ Custom Transposome Construction Kit with pMOD-2 VectorTNP10622 20 reactions

EZ-Tn5™ Custom Transposome Construction Kit with pMOD-3 VectorTNP10623 20 reactions

TypeOne™ Restriction InhibitorIncrease transformation efficiency of most bacterial strains

DNA transformation can be difficult to achieve in many bacterial strains due to the presence of one or more restriction and modification (R-M) systems that cleave unmodified DNA that is recognized as “foreign.” TypeOne™ Restriction Inhibitor* provides a powerful method for increasing transformation efficiencies in bacterial strains with type I R-M systems. Type One Restriction Inhibitor also increases the insertion efficiency of EZ-Tn5 Transposome complexes. It has also been reported to block Type III restriction systems.

• Easy to use: Simply add TypeOne Restriction Inhibitor to unmodified DNA or a Transposome and electroporate using standard methods.

• Blocks all known families of type I R-M enzymes and can therefore be used to increase transformation efficiencies in a variety of bacterial cells.

*Covered by issued and/or pending patents. See www.epicentre.com/legal.

Cat.# Quantity

TypeOne™ Restriction InhibitorTY0261H 100 µg

techhelp@epicentre.com • (800) 284-8474 5

In Vivo Transposomics™ Research

Figure1.OverviewoftheprocedurefortheEZ-Tn5™CustomTransposome™ConstructionKit.

Target DNA

Transposon Transposase

In Vitro Use

In Vivo Use

EZ-Tn5™Transposon

EZ-Tn5™Transposome™

EZ-Tn5™Transposome™

Cells

1. Electroporate2. Plate

Insertion Clones

Phenotypic Analysis

Direct GenomicDNA Sequencing

Rescue Cloning

10 min, 37°C

Selection Plate

1. Incubate 37°C; 2 hrs2. Transform E. coli3. Select Drugr clones

pMOD™-3<R6Kγori/MCS>

ForwardPCR Primer

ForwardSequencing

PrimerReverse

PCR Primer

ME MEPvu IIPshAI

Pvu IIPshAI

pUC-19 MultipleCloning SiteEcoRI HindIII

R6Kγ ori

ReverseSequencing

Primer

Vector( )colE1 ori, AmpR

pMOD™-2<MCS>

ForwardPCR Primer

ForwardSequencing

PrimerReverse

PCR Primer

ME MEPvu IIPshAI

Pvu IIPshAI

pUC-19 MultipleCloning SiteEcoRI HindIII

ReverseSequencing

Primer

Vector( )colE1 ori, AmpR

4. Prepare template DNA

minus Mg++

MEME

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In Vitro Transposon Insertion Kits

EZ-Tn5™ <KAN-2>, EZ-Tn5™ <TET-1>, and EZ-Tn5™ <DHFR-1> Insertion KitsCompletely sequence clones without subcloning or primer walking

EZ-Tn5 Insertion Kits* are designed to simplify and speed up complete sequencing of any cloned DNA >2 kb, without primer walking or subcloning. A simple, one-step in vitro reaction results in random insertion of a single EZ-Tn5 Transposon containing a selectable marker into the clone. Then, transform E. coli cells with an aliquot of the reaction and select for the marker encoded by the EZ-Tn5 Transposon. Use the primers provided in the kits to sequence insertion clones bidirectionally from primer binding sites at the ends of the inserted transposon.

• Primer binding sites are distributed throughout the clone, ensuring much better sequence coverage compared to primer walking or subcloning.

• A single reaction generates up to 106 insertion clones—enough to sequence even the largest clone. Save time usually spent subcloning or designing and synthesizing sequencing primers.

• Use a single set of sequencing primers (provided in the kits) to completely sequence any clone.

• The EZ-Tn5 System can be used for processing multiple DNA samples, making it an effective component of a high-throughput sequencing pipeline.

*Covered by intellectual property rights licensed to Epicentre.

Cat.# Quantity

EZ-Tn5™ <KAN-2> Insertion Kit EZI982K 10 Reactions

EZ-Tn5™ <TET-1> Insertion Kit EZI921T 10 Reactions

EZ-Tn5™ <DHFR-1> Insertion Kit EZI912D 10 Reactions

Contents: EZ-Tn5 Transposase, EZ-Tn5 Transposon, EZ-Tn5 10X Reaction Buffer, EZ-Tn5 10X Stop Solution, Forward and Reverse Sequencing Primers, Control Target DNA, Sterile Water.

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In Vitro Transposon Insertion Kits

EZ-Tn5™ <oriV/KAN-2> Insertion Kit The EZ-Tn5 <oriV/KAN-2> Insertion Kit* randomly inserts a single EZ-Tn5™ <oriV/KAN-2> Transposon containing an inducible, high-copy origin of replication (oriV) into the target DNA. Thus, the kit integrates CopyControl™ capability into those low- to single-copy-number clones (such as BACs or fosmids) that do not possess this feature. Selected clones are amplified by adding the CopyControl Induction or Autoinduction Solution, and microgram quantities of template DNA can be purified from as little as 1 ml of culture.

EZ-Tn5™ <T7/KAN-2> Promoter Insertion KitThe EZ-Tn5 <T7/KAN-2> Promoter Insertion Kit* provides an easy and reliable method to randomly insert a transposon containing a phage T7 RNA polymerase promoter into any DNA molecule in vitro.

EZ-Tn5™ <R6Kγori/KAN-2> Insertion KitThe EZ-Tn5 <R6Kγori/KAN-2> Insertion Kit* facilitates the sequencing and genetic analysis of plasmids or any other circularized DNA that would not otherwise replicate in E. coli. The kit is based upon the EZ-Tn5 <R6Kγori/KAN-2> Transposon, which carries the E. coli R6Kγ conditional origin of replication (R6Kγori) and a kanamycin- resistance marker. Clones can be maintained in Epicentre’s TransforMax™ EC100D™ pir+ or TransforMax™ EC100D™ pir-116 strains.*Covered by intellectual property rights licensed to Epicentre.

Cat.# Quantity

EZ-Tn5™ <oriV/KAN-2> Insertion KitEZI02VK 10 ReactionsContents: EZ-Tn5 Transposase, EZ-Tn5 <oriV/KAN-2> Transposon, EZ-Tn5 10X Reaction Buffer, EZ-Tn5 10X Stop Solution, Forward and Reverse Primers, Control Target DNA, Sterile Water.

EZ-Tn5™ <T7/KAN-2> Promoter Insertion KitEZI03T7 10 ReactionsContents: EZ-Tn5 Transposase, EZ-Tn5 <T7/KAN-2> Transposon, EZ-Tn5 10X Reaction Buffer, EZ-Tn5 10X Stop Solution, Forward and Reverse Primers, Control Target DNA, Sterile Water.

EZ-Tn5™ <R6Kγori/KAN-2> Insertion KitEZI011RK 10 ReactionsContents: EZ-Tn5 Transposase, EZ-Tn5 <R6Kγori/KAN-2> Transposon, EZ-Tn5 10X Reaction Buffer, EZ-Tn5 10X Stop Solution, Forward and Reverse Primers, Control Target DNA, Sterile Water.

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DNA Purification

Cat.# Quantity

Meta-G-Nome™ DNA Isolation Kit MGN0910 10 PurificationsContents: Extraction buffer, Filter Wash Buffer, Meta-Lysis Solution (2X), MPC Protein Precipitation Reagent, Proteinase K, Fosmid Control DNA, RNase A, Ready-Lyse Lysozyme Solution, TE Buffer, 0.45-µm cellulose acetate filter membranes, 1.2-µm cellulose acetate filter membranes.

Meta-G-Nome™ DNA Isolation KitPurify metagenomic DNA from water or soil samples

The Meta-G-Nome DNA Isolation Kit is used to isolate DNA from unculturable or difficult-to-culture microbial species present in environmental water or soil samples. The simple extraction procedure involves low-speed centrifugation prior to filtration, and lysis with Ready-Lyse™ Lysozyme and Proteinase K. 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. The kit can also be used to prepare 16S rRNA metagenomic libraries for phylogenetic classification or species-abundance studies.

• Isolated DNA is also suitable for PCR (Fig. 1) or sequencing.• No bead-beating, agarose plugs, pulse-field gel electrophoresis, or size selection.• No phenol/chloroform extractions or CTAB.

M 1 2 3 4 5 6DNA 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.

Figure1.PCRamplificationofsoilmetagenomicDNA.

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DNA Purification

MasterPure™ Complete DNA and RNA Purification KitPurify TNA, DNA, or RNA from any source

The MasterPure Complete DNA and RNA Purification Kit enables efficient purification of intact total nucleic acid (TNA), DNA, or RNA from every type of biological material. The purified DNA is suitable for sequencing and other molecular biology applications.

• Purify DNA in 30 minutes without spin columns.• A260/A280 ratios consistently between 1.8 and 2.0.• Recover >90% of theoretical DNA yield, including both low- and high-molecular-

weight nucleic acids.• No phenol, chloroform, or other caustic solvents.

Cat.# Quantity

MasterPure™ Complete DNA and RNA Purification KitMC89010 5/10 PurificationsMC85200 200 PurificationsFor isolating TNA, DNA, or RNA.Contents: Red Cell Lysis Solution, Tissue and Cell Lysis Solution, MPC Protein Precipitation Reagent, 2X T&C Lysis Solution, TE Buffer, RNase A, RNase-Free DNase I, Proteinase K, 1X DNase Buffer, RiboGuard RNase Inhibitor.Note: Cat. # MC89010 contains reagents for 10 TNA, 10 DNA, or 5 RNA purifications and Cat. # MC85200 contains reagents for 200 TNA, 200 DNA, or 100 RNA purifications.

Sample SampleSize TNAµg DNAµg RNAµgHeLa/HL60 cells 1 x 106 cells 10-30 3-12 7-15TissuesLiver 5 mg 33-42 5-10 13-25Brain 5 mg 9-13 6-9 4-11Heart 5 mg 6-10 4-7 4-5Kidney 5 mg 10-17 3-8 14-17Thymus 5 mg 15-30 6-12 9-18Blood 200 µl 3-10 3-9Buffy coat 300 µl 40-55 40-55 3-6Mouse tail 0.5 cm 25-30 9-11  E. coli 3.5 x 106 cells 2.5-2.8 1.3-1.6 1.6-1.8S. mutans 1.5 ml 0.9    Yeast* 2.2 x 106 cells     11-18(S. cerevisiae) 1.1 x 107 cells     70-78

*For extraction of yeast DNA, the MasterPure™ Yeast DNA Purification Kit is recommended.

Table1.TypicalyieldsobtainedusingtheMasterPure™CompleteKit.

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DNA Purification

MasterPure™ Gram-Positive DNA Purification KitPurify genomic DNA from Gram-positive bacteria

The MasterPure Gram-Positive DNA Purification Kit provides all of the reagents needed to purify DNA from Gram-positive bacteria. The purified DNA is suitable for fosmid library construction, PCR, restriction digests, Southern blotting, and other molecular biology applications. The protocol can also be adapted for total RNA purification.

• Scalable method for large sample volumes.• Yields high-quality, high-molecular-weight DNA.• Lysozyme included; no separate purchase required.• Tested on a variety of species.• Can also be used with Gram-negative bacteria.

Cat.# Quantity

MasterPure™ Gram-Positive DNA Purification KitMGP04020 20 ReactionsMGP04100 100 Reactions

Contents: Gram-Positive Cell Lysis Solution, MPC Protein Precipitation Reagent, Ready-Lyse

Lysozyme, Proteinase K, TE Buffer, RNase 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.

12 kb

1 kb

M 1

Figure1.ElectrophoreticanalysisofDNApurifiedusingtheMasterPure™Gram-PositiveDNAPurificationKit.

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DNA Purification

MasterPure™ Yeast DNA Purification KitPurify DNA at high yields from yeast

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. The protocol can be easily scaled for larger or smaller samples, including single yeast colonies. The recovered nucleic acid can be used directly in most applications, including sequencing, PCR amplification, restriction endonuclease digestion, Southern blotting, and genomic library preparation.

• Higher yields of yeast chromosomal DNA than with other kits (Fig. 1).• Recover DNA from a wide variety of yeast species, including Candida, Saccharomyces,

Pichia, and Schizosaccharomyces, and filamentous fungi such as Aspergillus and Penicillium.

• Purify high-molecular-weight DNA in less than 40 minutes.• No bead-beating, columns, or phenol or other organic extractions.

Cat.# Quantity

MasterPure™ Yeast DNA Purification KitMPY80010 10 PurificationsMPY80200 200 PurificationsContents: Yeast Cell Lysis Solution, MPC Protein Precipitation Reagent, TE Buffer, RNase A.

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. cerevisiae than other kits.

0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000DNA, nanograms

MasterPure™ Kit – Supplier Q – Supplier F – MasterPure™ Kit –

Supplier Q – Supplier F –

C. albicans

S. cerevisiae

Figure1.ComparativeyieldsofDNA.

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RNA Purification

MasterPure™ Yeast RNA Purification KitPurify RNA from yeast and filamentous fungi

The MasterPure Yeast RNA Purification Kit provides all of the reagents needed to purify RNA from yeast cell types (including Candida, Saccharomyces, Schizosaccharomyces, Pichia) and filamentous fungi. The kit uses a rapid salting-out process to remove contaminating macromolecules. The purified RNA is suitable for cDNA synthesis and microarray gene expression analysis.

• No hot acid phenol, spin columns, or bead-beating.• Faster than spheroplasting.• No extra enzymes or equipment to purchase.• Yields 25-50 µg RNA from 1 ml of mid-log S. cerevisiae.

Cat.# Quantity

MasterPure™ Yeast RNA Purification KitMPY03010 10 ReactionsMPY03100 100 ReactionsContents: Extraction Reagent for RNA, MPC Protein Precipitation Reagent, TE Buffer (in 100-reaction kit only), Proteinase K, RNase-Free DNase I, RiboGuard RNase Inhibitor, 10X DNase Buffer, 2X T & C Lysis Solution.

RNA was extracted from S. cerevisiae using the MasterPure™ Kit and stored at –20°C for 2 months before analysis on an Agilent Technologies 2100 Bioanalyzer. The electropherogram demonstrates the high-purity, intact RNA.

Figure1.PurityofRNA.

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DNA Extraction

QuickExtract™ Bacterial DNA Extraction KitExtract DNA from Gram-positive and Gram-negative Bacteria

The QuickExtract Bacterial DNA Extraction Kit provides a simple method for extracting DNA from Gram-positive and Gram-negative bacteria. The kit contains QuickExtract Bacterial DNA Extraction Solution and Ready-Lyse™ Lysozyme Solution, a proven reagent with over 200 times the specific activity of egg-white lysozyme. After mixing and incubating for 15 minutes, the isolated DNA is ready for PCR or other downstream applications such as restriction digests, PFGE (pulsed-field gel electrophoresis), and optical mapping. The kit has been tested on a range of bacteria (Table 1) and has been shown to produce very long DNA strands (Fig. 1).

• Fast—PCR-ready DNA in 15 minutes, ideal for high-throughput workflows.• Safe—No organic extraction.• Efficient—No columns, transfers, or sample loss.

Cat.# Quantity

QuickExtract™ Bacterial DNA Extraction KitQEB0905T 5 ml (50 Extractions)QEB09050 50 ml (500 Extractions)

Contents: QuickExtract Bacterial DNA Extraction Solution, Ready-Lyse Lysozyme Solution.

Gram-positive Gram-negativeBacillus subtilis E. coliBifidobacterium spp Salmonella entericaBrevibacterium linens Salmonella typhimuriumClostridium perfringens Vibrio gazogenesLactobacillus plantarum Listeria monocytogenes Staphylococcus equorum Streptococcus agalactiae Streptococcus pyogenes Streptococcus thermophilus

Table1.SpeciesofbacteriathathavebeentestedwiththeQuickExtract™BacterialDNAExtractionKit.

Many strands are 400-500 kb. Image courtesy of Dr. Shiguo Zhou, University of Wisconsin-Madison.

~250 kb

Figure1.LongDNAstrandsfromE. coliextractedusingtheQuickExtract™BacterialDNAExtractionKit.

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DNA-Seq

End-It™ DNA End-Repair KitRepair DNA ends for adaptor ligation

The End-It DNA End-Repair Kit converts DNA containing damaged or incompatible 5′- and/or 3′-protruding ends to 5′-phosphorylated, blunt-ended DNA. The end-repaired DNA can be used for ligation into a cloning vector or ligation of next-generation sequencing adaptors, using the Fast-Link™ DNA Ligation Kit. The high specific activity of the End-Repair Enzyme Mix provides complete conversion of protruding ends to 5′-phosphorylated, blunt-ended DNA.

The End-It DNA End-Repair Kit contains reagents for 20 or 50 end-repair reactions (repair of up to 100 µg or 250 µg of genomic DNA, respectively).

Cat.# Quantity

End-It™ DNA End-Repair KitER0720 20 ReactionsER81050 50 ReactionsContents: End-Repair Enzyme Mix, End-Repair 10X Buffer, dNTP Solution, ATP.

Fast-Link™ DNA Ligation KitLigate next-generation sequencing adaptors to DNA

The Fast-Link DNA Ligation Kit uses a high-quality ligase (Fast-Link T4 DNA Ligase), to provide extremely rapid, high-efficiency DNA ligation. Cohesive-end ligations can be performed in 5 minutes at room temperature. In contrast to other ligases, it is not necessary to desalt Fast-Link ligation reactions prior to transformation of electrocompetent or chemically competent cells. The Fast-Link Kit can be used for routine and high-throughput DNA cloning.

Cat.# Quantity

Fast-Link™ DNA Ligation KitLK11025 25 LigationsLK0750H 50 LigationsLK6201H 100 LigationsContents: Fast-Link DNA Ligase, Fast-Link 10X Ligation Buffer, 10 mM ATP.

techhelp@epicentre.com • (800) 284-8474 15

DNA-Seq

CircLigase™ and CircLigase II ssDNA LigasesCircularize ssDNA templates

CircLigase ssDNA Ligase* is a thermostable ATP-dependent ligase that catalyzes intramolecular ligation (i.e., circularization) of ssDNA templates having a 5′-phosphate and a 3′-hydroxyl group. In contrast to other ligases, CircLigase ssDNA Ligase ligates ends of ssDNA in the absence of a complementary sequence. The enzyme is therefore useful for making circular ssDNA molecules from linear ssDNA. Circular ssDNA molecules can be used as substrates for rolling-circle replication or rolling-circle transcription.

CircLigase II ssDNA Ligase* has greater activity and a new Reaction Buffer for improved ligation efficiency.

Unit Definition: One unit of CircLigase enzyme converts 1 pmol of a linear 5′-phosphorylated CircLigase Standard 55-mer Oligo into an Exonuclease I–resistant circular form in 1 hour at 60°C under standard assay conditions.*Covered by intellectual property rights licensed to Epicentre.

Cat.# Quantity

CircLigase™ ssDNA LigaseCL4111K 1,000 Units CL4115K 5,000 UnitsContents: CircLigase ssDNA Ligase, CircLigase 10X Reaction Buffer, 1 mM ATP, 50 mM MnCl2, CircLigase ssDNA Control Oligo, Sterile Water.

CircLigase™ II ssDNA LigaseCL9021K 1,000 Units CL9025K 5,000 UnitsContents: CircLigase II ssDNA Ligase, CircLigase II 10X Reaction Buffer, 50 mM MnCl2, CircLigase ssDNA Control Oligo, Betaine, Sterile Water.

DisplaceAce™ DNA Polymerase Improve DNA sequencing of GC-rich regions

DisplaceAce DNA Polymerase* is a recombinant DNA polymerase modified to remove 5′→3′ exonuclease activity. It has strong strand-displacing DNA polymerase activity, similar to that of Bacillus DNA polymerases. The DNA-dependent DNA polymerase activity is optimal at approximately 65°C. It also has RNA-dependent DNA polymerase activity. The enzyme can be inactivated by incubation at 80°C for 20 minutes.

Unit Definition: One unit converts 10 nmol of dNTPs into acid-insoluble material in 30 minutes at 65°C under standard assay conditions.*Covered by issued and/or pending patents.

Cat.# Concentration Quantity

DisplaceAce™ DNA PolymeraseD090710K 100U/ul 10,000 Units

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rRNA Removal

RiboZero™ rRNA Removal KitsRemove up to 99.9% of the rRNA from a total RNA sample

The Ribo-Zero™ rRNA Removal Kits remove more ribosomal RNA (rRNA) from total RNA preparations than any other method. The kits are ideal for preparing RNA samples for whole-transcriptome RNA-Seq, random-primed cDNA synthesis, and other RNA analysis applications.

• Suitable for both intact and partially degraded RNA.• Recovers mRNA and small and large ncRNAs for whole-transcriptome RNA-Seq.• Single-pass, 90- to 120-minute process.• Recovers all fragments of nonribosomal RNAs in a partially degraded RNA sample.

Cat.# Quantity

Ribo-Zero™ rRNA Removal Kit (Meta-Bacteria)RZMB11086 6 Reactions

Ribo-Zero™ rRNA Removal Kit (Gram-Negative Bacteria)RZNB1056 6 Reactions

Ribo-Zero™ rRNA Removal Kit (Gram-Positive Bacteria)RZPB10106 6 Reactions

InputRNA

PercentRemovalofrRNA

23S 16S 5S

Partially degraded B. subtilis total RNA

>99.9% >99.9% 93.5%

Intact E. coli total RNA >99.9% >99.9% >97%

Table1.RNAremovalwithRibo-Zero™KitsdeterminedbyqPCR.

techhelp@epicentre.com • (800) 284-8474 17

RNA-Seq

Cat.# Quantity

ScriptSeq™ mRNA-Seq Library Preparation Kit (Illumina®-Compatible)SS10906 6 ReactionsSS10924 24 Reactions

RNA-Seq Barcode Primers (Illumina®-Compatible)RSBC10948 48 Reactions eachSet of 12 Illumina®-compatible barcodes.

ScriptSeq™ mRNA-Seq Library Preparation KitsDirectional mRNA-Seq libraries in 3 hours

The ScriptSeq mRNA-Seq Library Preparation Kits* use a unique terminal-tagging technology* that simplifies the preparation of directional, paired-end libraries for Illumina® sequencing. The kits are suitable for any animal, plant, or bacterial species.

• Start with 50 ng or less of rRNA-depleted or poly(A)-enriched RNA.• Cluster-ready, directional libraries in about 3 hours without adaptor ligation.• Use Illumina-compatible barcodes (available separately) or user-defined barcodes.• Libraries demonstrate high concordance with MAQC qPCR data.• High percentage of mapped reads from intact, partially degraded, and FFPE RNA

samples.*Covered by issued and/or pending patents.

A. Intact UHRR and BrRR

Correlation between data obtained from ScriptSeq™ libraries and corresponding MAQC qPCR data. A) Libraries prepared from rRNA-depleted, intact UHRR and BrRR. B) Libraries prepared from partially fragmented, rRNA-depleted UHRR and BrRR. UHRR, Universal Human Reference RNA; BrRR, Brain Reference RNA.

Figure1.Correlationofgeneexpression.

B. Fragmented UHRR and BrRR

18 www.epicentre.com

Cloning

CopyControl™ Fosmid Library Production KitSave time and effort constructing a fosmid library

The CopyControl Fosmid Library Production Kits* clone randomly sheared DNA to produce unbiased genomic libraries containing 35- to 40-kb inserts. The unique CopyControl technology enables you to grow clones at single-copy number to ensure insert stability and cloning of expressed toxic sequences, and then induce the clones to high-copy number (20-50 copies per cell) for high yields of DNA.

• Kits include all the components required to construct 10 complete libraries. • Random shearing of genomic DNA ensures more complete and unbiased libraries

compared to partial restriction digestion.*Covered by issued and/or pending patents.

Cat.# Quantity

CopyControl™ Fosmid Library Production KitCCFOS110 1 KitComplete kit for constructing a CopyControl Fosmid library in the Cloning-Ready pCC1FOS Vector.

CopyControl™ HTP Fosmid Library Production KitCCFOS059 1 KitComplete kit for constructing a CopyControl Fosmid library in the Cloning-Ready pCC2FOS Vector.

Once the library has been prepared, individual clones can be cultured in small volumes and induced to multiple-copy number for high yields of high-purity DNA using Epicentre’s DirectLysis Fosmid96 Kit or FosmidMAX™ DNA Purification Kit.

Figure1.OverviewoftheprocessfortheCopyControl™Kits.

techhelp@epicentre.com • (800) 284-8474 19

Other Reagents

Ready-Lyse™ Lysozyme Solution Purify nucleic acids or protein from bacteria

Ready-Lyse™ Lysozyme Solution is a recombinant lysozyme preparation, derived from a nonmammalian, nonavian source, that is useful for the lysis of Gram-negative and Gram-positive bacteria. The specific activity of Ready-Lyse Lysozyme is 200-fold higher than that of egg-white lysozyme and, therefore, less lysozyme is needed in a reaction. Unlike egg-white lysozyme, Ready-Lyse Lysozyme Solution is stable at –20°C, eliminating the need to prepare a fresh solution for each use.

Unit Definition: One unit of Ready-Lyse Lysozyme produces a decrease in A350 of 0.001 per minute at 23°C with a 0.5 mg/ml suspension of lyophilized E. coli K802 cells in 50 mM Tris-HCl (pH 7.5).

Cat.# Quantity

Ready-Lyse™ Lysozyme SolutionR1802M 2 X 106 Units R1804M 4 X 106 UnitsR1810M 10 X 106 Units

Proteinase K Remove protein from nucleic acid preps

Proteinase K is used to digest protein and remove contamination from preparations of nucleic acid. The addition of Proteinase K to nucleic acid preparations rapidly inactivates nucleases that might otherwise degrade the DNA or RNA during purification. Proteinase K remains active under denaturing conditions, such as the presence of SDS or urea, chelating agents (EDTA, sulfhydryl reagents), and trypsin or chymotrypsin inhibitors.

Cat.# Concentration Quantity

Proteinase KMPRK092 50 µg/µl 2 ml

726

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I 537

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