Enzymes and Kits for Molecular Biology

CRISPR/Cas9 genome editing complex

EPICAT001, Rev A Table of Contents 3 Epicentre (an Illumina company) Enzymes 4-8 Properties of the Enzymes 9, 10 DNA Polymerases 11, 12 PCR Enzymes (Thermostable DNA Polymerases) 13 RNA Polymerases 14-21 , Proteases, and Lysozymes 22, 23 DNA 24 RNA Ligases 25 26, 27 Kinases, Glucosyltransferases, and Deadenylases

28 Reverse Transcriptases

Molecular Biology Kits and Reagents 29 Sample Collection Swabs and Brushes 30-32 DNA Extraction for PCR and qPCR 33-37 DNA and RNA Purification for Sequencing, Cloning, etc 38-40 PCR 41, 42 In Vitro Transcription 43 Target Labeling for Expression Microarrays 44, 45 RNA Amplification 46-48 Genomic DNA Cloning 49-51 Bacteria Mutagenesis (Transposon Mutagenesis)

©2015 Illumina. All rights reserved. Ampligase, AmpliScribe, APex, Baseline-ZERO, BeadChip, Catch-All, CircLigase, CopyControl, DisplaceAce, DuraScribe, DuraScript, EasyLyse, EC100, End-It, EPI300, EpiScript, EZ-TN5, Fast-Link, FosmidMAX, FailSafe, HK, Hybridase, MasterAmp, MasterPure, MessageBOOSTER, Meta-G-Nome, OmniCleave, Plasmid-Safe, QuickExtract, Ready-Lyse, RepliPHI, RiboShredder, TargetAmp, Terminator, T7 R&DNA, T7-Flash TransforMax, Transposome, Transposomics are trademarks of Epicentre.

For Research Use Only. Not for use in diagnostic procedures

2 www.epicentre.com Enzymes

Enzymes

Expert purification and formulation Epicentre began manufacturing high-purity enzymes for molecular biology in 1987 and continues to do so at a state-of-the-art facility in Madison, Wisconsin, USA. Our enzyme portfolio includes both unique, hard-to-find enzymes and standard ligases, kinases, and polymerases.

Quality you can depend on • High purity and high activity • Stringent quality control • ISO 13485 compliant • Lot-to-lot consistency

Bulk and OEM opportunities Many of the enzymes are available in bulk quantity and for OEM sales. Please inquire.

Unique enzymes and products What will you create using Epicentre enzymes • CircLigase™ DNA – convert linear ssDNA into circular DNA • Plasmid-Safe DNase – remove linear dsDNA from closed circular DNA • R – enrich for circular RNAs • Ampligase™ DNA Ligase – thermostable DNA ligase • EpiScript™ RNase H- Reverse Transcriptase – great value on an RNAse H- RT • Quick Extract™ DNA Extraction Solution – PCR-ready DNA in 8 minutes

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 3 Enzyme Properties

Mesophilic DNA Polymerases Activity 5′→3′ 3′→5′ Strand Product Name exonuclease Heat Inactivationa displacement RepliPHI Phi29 DNA Polymerase – ++ 65°C for 10 minutes ++++ DNA Polymerase I, E. coli + ++ 75°C for 20 minutes – Klenow DNA Polymerase – ++ 75°C for 20 minutes + Exo-Minus Klenow DNA Polymerase (D355A, E357A) – – 75°C for 20 minutes + T4 DNA Polymerase – +++ 75°C for 20 minutes – Terminal deoxynucleotidyl , Recombinant – – 75°C for 20 minutes – a Indicated treatment results in complete inactivation under standard reaction conditions.

Thermophilic DNA Polymerases Activity 5′→3′ 3′→5′ Product Name exonuclease exonuclease Thermostabilitya Fidelityb MasterAmp – – N/A N/A AmpliTherm DNA Polymerase MasterAmp Taq DNA Polymerase + – 97.5°C for 9 minutes 0.38-1.82 x 104 MasterAmp Tth DNA Polymerase + – 95°C for 20 minutes 2.2 x 104 DisplaceAce DNA Polymerase – – 65°C for 30 minutes N/A a Values represent half-lives: 50% of the enzymatic activity is retained after the given time at the stated temperature. b Defined as the average number of correct a polymerase incorporates before making an error.

RNA Polymerases Activity 5′→3′ 3′→5′ Product Name exonuclease exonuclease Heat Activation T7 RNA Polymerase – – 70°C for 10 minutes E. coli RNA Polymerase Core Enzyme – – 75°C for 10 minutes T7 R&DNA Polymerase – – 70°C for 10 minutes Poly(A) Polymerase – – 65°C for 20 minutes

For Research Use Only. Not for use in diagnostic procedures

4 www.epicentre.com Enzyme Properties

DNA Heat Enzyme Substrate Activity Products Applications Inactivation

Baseline-ZERO dsDNA Digests dsDNA or ssDNA to 75°C for Mononucleotides DNA removal from RNA prep DNase and ssDNA mononucleotides 20 minutes

Activated by divalent cations. In presence of Mg2+, it cleaves each DNA strand randomly and independently, preferentially DNA removal from RNA prep. dsDNA Oligos and dNMPs with 5′ 75°C for RNAse-Free DNase I adjacent to pyrimidines. In Random dsDNA nicking. and ssDNA phosphate and 3′ OH. 20 minutes presence of Mn2+, it cleaves both DNase footprinting. strands simultaneously, generating fragments with blunt ends or 1-2- base overhangs. DNA repair and anti-tumor dsDNA with single- IV dsDNA with Cleaves sugar-phosphate bond 5′ drug research. Base Excision gaps. The cleaved ssDNA N/A (E. coli) abasic site of an abasic site. Sequence Scanning of DNA strand has a 3′ OH. containing dUMPs.

UV-irradiated First cleaves N-glycosidic bond 5′ Nicked dsDNA with an abasic DNA with of thymine dimers, then cleaves site at the 3′ end of the cut and Research on repair of DNA T4 Endonuclease V N/A thymine sugar-phosphate bond 3′ of the thymine-dimer bases at the exposed to UV light. dimers abasic site. 5′ end of the cut. Rapid sizing or restriction dsDNA with Cleaves both strands at 5′ ends with overhangs Lambda Terminase mapping of BAC, fosmid or N/A cos sites bacteriophage lambda cos sites. 12 bases in length. cosmid clones. Cleaves dsDNA with Pvu Rts 1I 3′ ends with overhangs Analyzing 5-hmC patterns dsDNA 5-hydroxymethylcytosines N/A Endonuclease 2 bases in length. within genome. (5-hmC).

DNA Heat Enzyme Substrate Activity Products Applications Inactivation Exonuclease I Removal of ssDNA and 80°C for ssDNA 3′→ 5′ exonuclease dNMPs (E. coli) oligonucleotides. 15 minutes Use with S1 or Mung 3′→ 5′ exonuclease digests duplex dNMPs and ssDNA on Bean Nuclease to make nested DNA from the 3′-end of a nick or a the opposite strand. deletions. Prepare ssDNA Exonuclease III blunt or 3′-recessed end; not active on Partial digestion 70°C for dsDNA templates for sequencing. (E. coli) thionucleotides. Exo III also has RNase H, produces dsDNA 20 minutes Site-directed mutagenesis. 3′-DNA and apurinic DNA having 5′ extensions Preparation of labeled endonuclease activities. of ssDNA. strand-specific probes.

Exonuclease that digests in both 5′→3′ Removae primers and 95ºC for Exonuclease VII ssDNA dNMPs and 3′→ 5′ directions. single-stranded oligos. 10 minutes dNMPs and ssDNA on 5′→3′ exonuclease digests dsDNA from the opposite strand. Lambda 5′-phosphorylated blunt or recessed ends. Partial digestion Prepare ssDNA templates for 75°C for dsDNA Exonuclease It has low activity on 5′-hydroxylated ends produces dsDNA sequencing. 10 minutes and is not active on nicked DNA. having 3′ extensions of ssDNA. An ATP- and Mg2+ dependent exonuclease RecBCD dsDNA and that digests linear DNA in both 5′→ 3′ and Remove linear DNA from circular Nuclease dNMPs N/A ssDNA 3′→ 5′ directions. Not active on nicked or DNA. (E. coli) closed-circular dsDNA.

Rec J 5′→ 3′ exonuclease digests ssDNA with a Remove primers and ssDNA from 65°C for ssDNA dNMPs Exonuclease 5′-phosphate or a 5′-OH. dsDNA. 20 minutes 5′→ 3′ exonuclease with single-strand- dNMPs. Circular ssDNA Plasmid mutagenesis. specific endonuclease activity in presence is obtained from Oligonucleotide site-directed dsDNA and of 1-10 mM Mg2+ ions. At <1 mM Mg2+, the T5 Exonuclease closed-circular dsDNA mutagenesis. Linear DNA removal N/A ssDNA 5′→ 3′ exonuclease can digest from a nick in presence of <1 mM from plasmid preps. Prepare in closed-circular dsDNA Mg2+. circular ssDNA. without digesting the opposite strand. Plasmid-Safe linear Remove chromosomal DNA Selectively digests linear DNA. No activity ATP-Dependent ssDNA and dNMPs fragments from plasmid, fosmid, N/A on nicked or closed-circular dsDNA. DNase dsDNA and BAC preparations.

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 5 Enzyme Properties

RNA Endonucleases Heat Enzyme Substrate Activity Products Applications Inactivation Oligoribonucleotides with Remove RNA from DNA preps. Cleaves ssRNA 3′ of RNase A ssRNA 3′-cytidine or 3′-uridine RNase protection assays. N/Aa pyrimidine residues residues RNA mapping and structure studies. Remove RNA from DNA preps. NMPs with 5′-OH Cleaves ssRNA between all RNase protection assays. Mismatch 70°C for RNase I, E. coli ssRNA and 2′,3′-cyclic dinucleotide pairs detection of single basepairs in 15 minutes monophosphate RNA:RNA or RNA:DNA hybrids. Cleaves dsRNA in presence Randomly cleave long dsRNA to of Mg2+ to 12-15-bp dsRNA. dsRNA with short dsRNA. RNA interference (RNAi). RNase III, E. coli dsRNA Cleaves dsRNA in presence of 5′-phosphate and 2-base No Studies on RNA structure, 20 mM Co2+ or Mn2+ to 3′-overhangs with 3′-OH RNA processing and maturation. 18-25-bp dsRNA Eliminate RNA prior to RNA in Cleaves RNA in RNA:DNA Oligoribonucleotides with second strand cDNA synthesis. 65°C for RNase H, E. coli RNA:DNA hybrid without affecting 5′-phosphate and 3′-OH Remove. poly(A) tails from 10 minutes hybrid unhybridized RNA or DNA mRNA hybridized to oligo(dT)

Hybridase RNA in Cleaves RNA in RNA:DNA Oligoribonucleotides with Thermostable RNA:DNA hybrid without affecting High-stringency hybrid selection. No 5′ phosphate and 3′ OH RNase H hybrid unhybridized RNA or DNA

RNase T1, Oligoribonucleotides with RNA mapping and structure studies. Aspergillis ssRNA Cleaves ssRNA 3′ of GMPs N/Aa 3′-GMP residues Remove RNA from DNA preps. oryzae RiboShredder Remove all RNA from genomic ssRNA Efficiently degrades all RNA NMPs No RNase Blend and cloned DNA preps. a N/A = Not applicable

Nucleases Active on both DNA and RNA Heat Enzyme Substrate Activity Products Applications Inactivation Terminator 5′→3′ exonuclease digests ssDNA or Remove 5′-monophosphorylated DNA 5′-Phosphate- ssDNA or ssRNA with 5′-monophosphorylated or primers or oligos. 65°C for dNMPs or NMPs Dependent ssRNA ends, but not with 5′-hydroxyl, Enrich ssDNA or ssRNA molecules 10 minutes Exonuclease 5′-triphosphorylated, or 5′-capped ends lacking 5′-monophosphate groups ssDNA, Remove DNA and RNA from OmniCleave Endonuclease efficiently di-, tri-, and dsDNA, protein preps. Remove host No Endonuclease digests DNA and RNA tetra-nucleotides or RNA DNA from phage preps

For Research Use Only. Not for use in diagnostic procedures

6 www.epicentre.com Enzyme Properties

DNA and RNA Ligases Ligation Template Type of Ends Ligated Ligation Required to Heat Name of Ligase Temperature Ligate Blunt Cohesive Primary Application Inactivation 65°C for Fast-Link DNA Ligase 20°C ATP Noa Yes Yes Rapid Cloning 10 minutes 65°C for T4 DNA Ligase 4°C - 25°C ATP Noa Yes Yes Cloning 15 minutes 65°C for E. coli DNA Ligase 5°C - 20°C NAD Yes; DNA only Weak Activity Yes Make long cDNA 20 minutes Ampligase Thermostable Template-dependent 20°C - 95°C NAD Yes; DNA only No Yes No DNA Ligase ligation Ligates 100°C for CircLigase ssDNA Ligase 20°C - 65°C ATP No N/Ab Make ssDNA Circles ssDNA 5 minutes Not Ligates 100°C for CircLigase II ssDNA Ligase 20°C - 65°C No N/Ab Make ssDNA Circles Needed ssDNA 5 minutes

Ligates 100°C for CircLigase RNA Ligase 25°C - 65°C ATP No N/Ab Make RNA Circles ssRNA 5 minutes

Ligate DNA T4 RNA Ligase 2, Not 65°C for 25°C No Ligates RNA or RNA to Ligate RNA to RNA Deletion Mutant needed 20 minutes RNA

a These enzymes ligate blunt ends of dsDNA, but ligation is more efficient on a ligation template, which can be DNA or RNA. b N/A=Not applicable.

Phosphatase Heat Enzyme Substrate Activity Products Applications Inactivation Apex Heat-Labile Dephosphorylated DNA/ Dephosphorylate of DNA/ 70°C for dsDNA, dsRNA Removes 5′ phosphate RNA RNA substrates 5 minutes 5′-di or Removes γ and 5′-monophosphorylated Ligation-tagging, 5′-end RNA 5′ Polyphosphatase triphosphorylated N/A ß phosphates RNA structure RNA

Kinases Heat Enzyme Substrate Activity Products Applications Inactivation Catalyzes transfer of T4 Polynucleotide y-phosphate of ATP to Phosphorylated DNA, Add 5′-phosphate to DNA 70°C for DNA, RNA Kinase 5′ terminus of DNA RNA or RNA 5 minutes (ds/ss) or RNA (w/3′OH)

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 7 Enzyme Properties

Glucosyltransferases Heat Enzyme Substrate Activity Products Applications Inactivation Transfers glucose from uridine Glucosylate or T4 Beta- Beta-glucosyl- 65°C for dsDNA diphosphoglucose to 5-hydroxy- immunodetect glucosyltransferase 5hmC 10 minutes methylcytosine (5-hmC) 5-hmC DNA

Deadenylases Heat Enzyme Substrate Activity Products Applications Inactivation Hydrolyzes Activated DNA (Ap-pDNA), 5′-phosphorylated Deadenyle 5′ end 70°C for 5′ Deadenylase 5′-5′-pyrophosphate activated RNA (Ap-pRNA) DNA or RNA of DNA or RNA 20 minutes linkage

Reverse Transcriptases Heat Enzyme Substrate Activity Products Applications Inactivation MMLV High Performance 85°C for ssRNA, ssDNA Synthesizes first strand cDNA cDNA cDNA synthesis Reverse Transcriptase 10 minutes EpiScript RNase 85°C for ssRNA, ssDNA Synthesizes first strand cDNA cDNA cDNA synthesis H– Reverse Transcriptase 5 minutes MonsterScript 1st-Strand 90°C for ssRNA, ssDNA Synthesizes first strand cDNA cDNA cDNA synthesis cDNA Synthesis 5 minutes

DNA Glycosylases and Excision Mixes

Enzyme Substrate Activity Products Applications Uracil base and abasic DNA. Fragment dUMP-containing DNA. HK-UNG Thermolabile dsDNA or Abasic sites can subsequently Cleave DNA at site-specific dUMP sites. Removes uracil base Uracil-N-Glycosylase (UNG) ssDNA be cleaved by AP , Base Excision Sequence Scanning of from dUMP residues containing such as Endonuclease IV, DNA containing dUMP residues. (UNG is also called Uracil-DNA in DNA Glycosylase or UDG) dUMP or by treatment with heat or alkaline bases. dsDNA with single-nucleotide Fragment dUMP-containing DNA. dsDNA or gaps where dUMP residues Cleave DNA at site-specific dUMP sites. Uracil-DNA Excision Mix Cleaves sugar- ssDNA have been removed, or Base Excision Sequence Scanning of (HK-UNG and phosphate bond 5′ containing ssDNA strands with lengths DNA containing dUMP residues. Endonuclease IV) of dUMPs dUMP equal to distances between dUMP residues

For Research Use Only. Not for use in diagnostic procedures

8 www.epicentre.com DNA Polymerases

RepliPHI™ Phi29 DNA Polymerase Cat. # Concentration Quantity RepliPHI Phi29 DNA Polymerase (φ29 DNA Polymerase) is a highly RepliPHI Phi29 Reagent Set processive enzyme with exceptional strand displacement activity. RH031110 1,000 U/µl 10,000 U The enzyme also contains a 3′→5′ exonuclease activity that RH040210 100 U/µl 10,000 U enables proofreading capability. Its specific activity is 1 x 106 U/mg. RepliPHI Phi29 DNA Polymerase (enzyme only) PP031010 1,000 U/µl 10,000 U PP040110 100 U/µl 10,000 U

DNA Polymerase I, E. coli Cat. # Concentration Quantity This DNA-dependent DNA polymerase contains both 5′→3′ and DNA Polymerase I, E. coli 3′→5′ exonuclease activities. The 5′→3′ exonuclease activity DP081025K 10 U/µl 2,500 U enables the enzyme to use nicks and gaps in the DNA as starting points for labeling the DNA by nick translation. • Generate labeled DNA probes by nick translation • Second strand cDNA • Synthesize in vitro

Cat. # Concentration Quantity Exo-Minus Klenow DNA Polymerase (D355A, E357A) Exo-Minus Klenow DNA Polymerase (D355A, E357A) KL11101K 10 U/µl 1,000 U This DNA-dependent DNA polymerase lacks both the 5′→3′ and 3′→5′ exonuclease activities of E. coli DNA polymerase I from which it is derived. • Random primer l DNA labeling • Second-strand cDNA synthesis • Strand displacement amplification

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 9 DNA Polymerases

T4 DNA Polymerase Cat. # Concentration Quantity Contains both a template-directed DNA polymerase activity and a T4 DNA Polymerase potent 3′→5′ exonuclease activity. D0605H 5 U/µl 500 U • Convert 5′- and 3′-protruding DNA termini to blunt ends • Clont PCR fragments • Label 3′-termini of DNA molecules and synthesize strand-specific probes

Terminal deoxynucleotidyl Transferase, Cat. # Concentration Quantity Recombinant Terminal deoxynucleotidyl Transferase, Recombinant TDT11725K 20 U/µl 2,500 U This enzyme catalyzes the addition of mononucleotides to the 3′-OH terminus of DNA molecules. Only one dNTP is needed for polymerization, although any dNTP (and derivative) or any combination of dNTPs will serve as a substrate. Protruding, recessed, or blunt-ended ssDNA or dsDNA molecules will serve as a template. The enzyme does not have 5′- or 3′-exonuclease activity. • Add of homopolymer tails to the 3′-OH terminus of DNA molecules • Label of 3′ ends of DNA with modified nucleotides • TdT-dependent PCR

DisplaceAce™ DNA Polymerase Cat. # Concentration Quantity This recombinant DNA polymerase has been altered by truncation DisplaceAce DNA Polymerase to remove the 5′→3′ exonuclease activity of the full-length D090710K 100 U/µl 10,000 U enzyme. It has strong strand-displacing DNA polymerase activity. 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. Thus, if thermal denaturation of a DNA substrate is intended (>75°C), the enzyme must be added after this step to ensure activity. • Most applications that require a strand-displacing DNA polymerase for high-temperature DNA synthesis • Synthesize first-strand cDNA from primed RNA templates

MasterAmp™ Tth DNA Polymerase Cat. # Concentration Quantity This recombinant DNA enzyme from Thermus thermophilus MasterAmp Tth DNA Polymerase has DNA polymerase activities up to ~95°C. High reaction TTH72250 5 U/µl 250 U temperatures can reduce nonspecific priming and template For use with MasterAmp™ PCR PreMixes secondary structure. Provided with MasterAmp™ PCR Enhancer Technology. • Amplify DNA by PCR • Improved PCR reactions of DNA templates having a high degree of secondary structure

For Research Use Only. Not for use in diagnostic procedures

10 www.epicentre.com PCR Enzymes

FailSafe™ Enzyme Mix Cat. # Quantity The FailSafe Enzyme Mix is a unique blend of thermostable DNA FailSafe Enzyme Mix Only polymerase and a 3′→5′ proofreading exonuclease that is capable FSE51100 100 U of amplifying most difficult templates. The FailSafe enzyme is FSE5101K 1,000 U extremely sensitive and provides a high-fidelity PCR product up to 20 kb. (For longer amplifications we recommend Epicentre’s MasterAmp Extra-Long PCR System). • PCR and multiplex PCR • PCR of difficult templates • High-sensitivity PCR • Amplify any sequence up to 20 kb by PCR

Cat. # Concentration Quantity MasterAmp AmpliTherm™ DNA Polymerase MasterAmp AmpliTherm™ DNA Polymerase This proprietary recombinant thermostable DNA polymerase for AT72250 5 U/µl 250 U use in PCR has optimal DNA synthetic activity at temperatures above 70°C and can be used at temperatures up to 95°C. It lacks For use with MasterAmp PCR PreMixes both the 5′→3′ structure-dependent exonuclease activity like that found in Taq DNA polymerase and the 3′→5′ proofreading exonuclease activity found in some other DNA polymerases. The enzyme is provided with the MasterAmp PCR Enhancer, which increases the probability of obtaining the desired amplification product and the reproducibility of PCR, and improves the consistency of PCR product yields in multiplex PCR. • Amplify DNA templates by PCR • Multiplex PCR

MasterAmp Taq DNA Polymerase Cat. # Concentration Quantity MasterAmp Taq DNA Polymerase Derived from Thermus aquaticus, this enzyme has optimal activity at temperatures above 70°C. It has an intrinsic 5′→3′ structure- Q82100 5 U/µl 100 U dependent exonuclease activity, but lacks 3′→5′ proofreading Q82500 5 U/µl 500 U exonuclease activity. Q8201K 5 U/µl 1,000 U Q8205K 5 U/µl 5,000 U The enzyme is provided with the MasterAmp PCR Enhancer, which increases the probability of obtaining the desired For use with MasterAmp PCR PreMixes amplification product and the reproducibility of PCR, and improves the consistency of PCR product yields in multiplex PCR. • PCR and multiplex PCR amplification of DNA templates

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 11 PCR Enzymes

MasterAmp 10X PCR Enhancer with Cat. # Quantity Betaine MasterAmp 10X PCR Enhancer with Betaine The MasterAmp 10X PCR Enhancer with betaine* substantially ME81205 5 ml improves PCR performance, and offers improved sensitivity and ME81210 10 ml specificity for amplification of many DNA templates, including GC-rich templates. Betaine reduces pauses/stops in GC-rich or other difficult regions of the template, thus increasing the yield of PCR products, and reducing the presence of nonspecific PCR products. • Simplifies PCR optimization • Enhances PCR yield and specificity • Compatible with most thermostable PCR enzymes *Covered by issued and/or pending patents

dNTP Solutions Cat. # Concentration Quantity • Sterile, neutral pH solutions of dATP, dCTP, dGTP, dTTP Premixed dNTP Solutions (All 4) • Available individually or as a PreMix D08104 2.5 mM 10 µmol (4 ml) D59104 25 mM 10 µmol (400 µl) dATP Solution D5910A 100 mM 10 µmol (100 µl) dCTP Solution D5910C 100 mM 10 µmol (100 µl) dGTP Solution D5910G 100 mM 10 µmol (100 µl) dTTP Solution D5910T 100 mM 10 µmol (100 µl) Also available: dUTP Solution D1905U 20 mM 5 µmol (250 µl)

For Research Use Only. Not for use in diagnostic procedures

12 www.epicentre.com RNA Polymerases

T7 RNA Polymerase Cat. # Concentration Quantity T7 RNA Polymerase produces defined RNA byin vitro transcription T7 RNA Polymerase of double-stranded DNA that is downstream of the respective RNA TM910K 200 U/µl 10,000 U polymerase promoter. TH950K 1,000 U/µl 50,000 U (Enzyme only. Transcription Buffer is not included.) • Extremely high promoter specificity For kits for in vitro transcription, please visit www.epicentre.com • Synthesize RNA for nucleic acid amplification methods or gene expression studies

T7 R&DNA™ Polymerase Cat. # Concentration Quantity A mutant form of T7 RNA polymerase (Y639F mutant). This T7 R&DNA Polymerase mutation enables T7 R&DNA Polymerase to incorporate D7P9201K 50 U/µl 1,000 U 2′-deoxyribonucleoside triphosphates (dNTPs) into full-length D7P9205K 50 U/µl 5,000 U RNAtranscripts much more efficiently than the corresponding wild- type T7 RNA Polymerase. This mutant enzyme uses the same T7 transcription promoters as the wild-type T7 RNA Pol. • Synthesize of RNA transcripts of mixed rNMP/2′-dNMP composition • Synthesize of modified RNA transcripts that are resistant to RNase A

E. coli RNA Polymerase Core Enzyme Cat. # Concentration Quantity Enzyme preparations are isolated from the rifampicin-sensitive E. coli RNA Polymerase Core Enzyme strain BL21. This is the only commercial purified Core Enzyme that C90100 1 U/µl 100 U has no detectable sigma subunit. C90500 1 U/µl 500 U

Poly(A) Polymerase Cat. # Concentration Quantity Adds a poly(A)- tail to the 3′-end of RNA. Poly(A) Polymerase Tailing Kit • Adds a poly(A) tail to RNA synthesized in vitro PAP5104H 4 U/µl 50 Reactions • Label the 3′ end of RNA with radioactive A residues

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 13 Nucleases, Proteases and Lysozymes

Baseline-ZERO™ DNase Cat. # Concentration Quantity Baseline-ZERO DNase digests dsDNA and ssDNA to Baseline-ZERO DNase mononucleotides more effectively than the commonly used bovine DB0711K 1 U/ul 1,000 MBU pancreatic DNase I. DB0715K 1 U/ul 5,000 MBU Following treatment with Baseline-ZERO, even the small DNA *Patent Pending oligonucleotides that remain after treatment with bovine pancreatic DNase I are undetectable. The enzyme provides a true zero baseline for RNA RT-PCR or microarray gene expression experiments. • Remove genomic DNA from RNA prior to RT-PCR or preparation of target RNA or cDNA for microarray analysis, especially for exon arrays or full coverage expression analysis • Remove small DNA oligonucleotides (e.g. random primers)

RNase-Free DNase Cat. # Concentration Quantity RNase-Free DNase I (bovine pancreas) is an endonuclease useful RNase-Free DNase I in removing DNA that might interfere with the characterization, D9905K 1 U/µl 5,000 MBU manipulation, or use of RNA; or for any application requiring highly D9910K 1 U/µl 10,000 MBU purified DNase I, such as nick translation. This enzyme efficiently hydrolyzes dsDNA and ssDNA to a mixture of short oligo- and mononucleotides. (Fig. 1) Figure 1. DNA removal from in vitro transcription reactions using RNase-Free DNase I. • Eliminate template DNA following in vitro synthesis of RNA with T7 phage RNA polymerase 1 2 3 4 • Label DNA by nick translation, in combination with Klenow or A linearized DNA template other DNA polymerases was transcribed using T7 RNA polymerase according • Treat RNA prior to RT-PCR —DNA to standard in vitro • Characterize DNA-protein interactions by DNase I footprinting transcription conditions. Lane 1, kb ladder; Lane 2, DNA control; Lane 3, transcription mixture; Lane 4, transcription mixture treated with 1 MBU —transcript of RNase-Free DNase I for 15 minutes at 37°C.

Endonuclease IV, E. coli Cat. # Concentration Quantity Endonuclease IV, cloned from the E. coli nfo gene, is a Endonuclease IV, E. coli metalloenzyme that functions in vivo to repair free radical damage in DNA. The enzyme also has Class II abasic endonuclease E70100 2 U/µl 100 U activity, which has utility in many areas of DNA damage and repair research. It is also useful in the study of the effects of anti-tumor drugs such as bleomycin on nucleic acids in vivo. • DNA repair research • Anti-tumor drug evaluation

For Research Use Only. Not for use in diagnostic procedures

14 www.epicentre.com Nucleases, Proteases and Lysozymes

T4 Endonuclease V Cat. # Concentration Quantity T4 Endonuclease V has N-glycosylase and apurinic/apyrimidinic T4 Endonuclease V (AP lyase) activities. T4 Endonuclease V binds to pyrimidine TE661K 20 U/µl 1,000 U dimers in dsDNA, then cleaves the N-glycosylic bond of the 5′-pyrimidine of the dimer (pyrimidine dimer DNA glycosylase) and breaks the phosphodiester bond 3′ to the resulting abasic site (3′ AP lyase). • Study UV damage to DNA and its repair • Detect differential UV damage repair of transcribed sequences • Detect UV mutational hotspots

Lambda Terminase Cat. # Concentration Quantity This endonuclease recognizes and cleaves DNA at cos sites, Lambda Terminase generating 5′ overhangs of 12 bases in length. Since the 12-base LT44200 2 U/µl 200 U cos site sequence is rare, the sizes of inserts in BAC, fosmid, or cosmid clones can be rapidly determined. The clones are linearized with lambda terminase and separated by pulsed field gel electrophoresis. Lambda Terminase can also be used for chromosomal mapping and for generating restriction maps of DNA cloned into BAC, cosmid, or fosmid vectors. • Rapidly size BAC, fosmid, or cosmid clones • Generati restriction maps of BAC, cosmid, or fosmid clone inserts • Linearize cos site-containing clones for in vitro packaging • Specifically cleave chromosomal DNA for physical mapping

Pvu Rts1I Endonuclease Catalog No. Concentration Size Pvu Rts1I is an endonuclease that cleaves dsDNA containing Pvu Rts1I Endonuclease 5-hydroxymethylcytosines (5-hmC). Under standard digestion RTS11100 1 U/μl 100 U conditions, the enzyme selectively cleaves DNA that contains the modified base on one or both strands. The consensus cleavage site of Pvu Rts1I is 5-hmCN11-12/N9-10G. • Differentiate 5-hmC from 5-mC • Cleave dsDNA at glucosylated and nonglucosylated 5-hmC

Exonuclease I, E. coli Cat. # Concentration Quantity Exonuclease I digests ssDNA in a 3′→ 5′ direction, but does not Exonuclease I, E. coli digest dsDNA. Although it requires the presence of magnesium X40505K 20 U/µl 5,000 U and a free 3′-hydroxyl terminus for activity, it is active under a wide X40520K 20 U/µl 20,000 U variety of buffer conditions and can be added directly into most reaction mixes. • Remove residual ssDNA, including oligos, from reaction mixes • Remove ssDNA from nucleic acid mixtures

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 15 Nucleases, Proteases and Lysozymes

Exonuclease III, E. coli Cat. # Concentration Quantity Exonuclease III digests duplex DNA in a 3′→ 5′ direction from Exonuclease III, E. coli a nick, a blunt end or 3′ recessed end, producing stretches of EX4425K 200 U/µl 25,000 U ssDNA on the opposite strand. • Produce intermediates for site-directed mutagenesis • Produce strand-specific radiolabeled probes

Exonuclease VII Cat. # Concentration Quantity Exonuclease VII has high enzymatic specificity for ssDNA and Exonuclease VII exhibits both 5′→ 3′ and 3′→ 5′ exonuclease activities. It is useful EN510250 10 U/µl 250 U for rapid removal of single-stranded oligonucleotide primers from a completed PCR reaction when different primers are required for subsequent PCR reactions. Exonuclease VII digestion of ssDNA occurs in the absence of magnesium. • Remove single-stranded oligonucleotide primers after PCR • Minimize the effect of primers left over from previous PCR reactions

Lambda Exonuclease Cat. # Concentration Quantity Lambda Exonuclease This highly processive 5′→ 3′ exodeoxy-ribonuclease selectively digests the phosphorylated strand of dsDNA. The preferred LE032K 10 U/µl 2,500 U substrate is blunt-ended, 5′-phosphorylated dsDNA. The enzyme has reduced activity against nicked DNA and against ssDNA and gapped DNA. • Analyze SSCP (single-strand conformation polymorphism) PCR products • Generate ssDNA sequencing template from PCR products

Figure 1. Lambda Exonuclease selectively digests the strand of a PCR product produced using a PCR primer with a 5′-phosphate.

Lambda Exonuclease

(5’-phosphate-specic 5’ 3’ )

Use the resulting single-stranded PCR product PCR using one primer for SSCP analysis or sequencing. with a 5’-phosphate

PCR product with strand-specic 5’phosphate

Treatment with Lambda Exonuclease

Single-stranded PCR product dNMPs

For Research Use Only. Not for use in diagnostic procedures

16 www.epicentre.com Nucleases, Proteases and Lysozymes

RecBCD Nuclease, E. coli Cat. # Concentration Quantity This exonuclease from E. coli degrades ssDNA and dsDNA. RecBCD Nuclease, E. coli Hydrolysis of the DNA is bi-directional from both the 3′ and 5′ BCD0401K 10 U/µl 1,000 U ends and processive, producing nucleoside monophosphates. Magnesium is required for the exonuclease activity, while calcium, nickel, zinc, and copper inhibit exonuclease activity. Calcium allows dsDNA unwinding (helicase activity) without hydrolysis. • Remove contaminating bacterial chromosomal DNA in plasmid, fosmid, cosmid, and BAC clone or vector preparations

Rec J Exonuclease Cat. # Concentration Quantity Rec J Exonuclease, derived from E. coli, catalyzes removal of Rec J Exonuclease deoxyribonucleoside monophosphates from ssDNA in a 5′→3′ RJ411250 10 U/µl 250 U direction. It's activity is dependent on Mg2+. Rec J Exonuclease can be heat-inactivated by incubation at 65°C for 20 minutes. • Remove primers from completed PCR reactions • Degrade single-stranded linear DNA in dsDNA and plasmid preps

Cat. # Concentration Quantity T5 Exonuclease T5 Exonuclease This highly efficient ′5 → 3′ exonuclease for either ssDNA T5E4111K 10 U/µl 1,000 U or ddDNA has a tightly associated single-strand-specific endonuclease activity when used in the presence of 1-10 mM divalent magnesium ions. Use low concentrations of magnesium ions (<1 mM) to selectively suppress this activity, allowing nicked, double-stranded circular DNA to be “gapped” to a single-stranded circular species. In the absence of divalent metal cofactors, T5 Exonuclease is able to bind to DNA with a single-strand arm adjacent to a duplex DNA region. • Plasmid mutagenesis methods • Oligonucleotide site-directed mutagenesis • Generate plasmid-sequencing templates • Remove denatured DNA from alkaline-based plasmid purification procedures for improved cloning procedures

Plasmid-Safe™ ATP-Dependent DNase Cat. # Concentration Quantity Plasmid-Safe ATP-Dependent DNase selectively removes Plasmid-Safe ATP-Dependent DNase contaminating bacterial chromosomal DNA from cosmid, BAC, E3101K 10 U/µl 1,000 U fosmid, and plasmid preparations. The enzyme will processively E3105K 10 U/µl 5,000 U degrade linear DNA from the ends; closed circular DNA (i.e. a E3110K 10 U/µl 10,000 U plasmid) does not have free ends, and is therefore not degraded. These properties make Plasmid-Safe ATP-Dependent DNase ideal for BAC and fosmid purification protocols such as shotgun sequencing and FISH where high purity DNA is necessary. • Remove contaminating bacterial chromosomal DNA in large- scale plasmid, cosmid, fosmid, BAC or vector preparations

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 17 Nucleases, Proteases and Lysozymes

RNase A Cat. # Concentration Quantity RNase A is an that cleaves ssRNA at the RNase A 3′ end of pyrimidine residues, forming oligoribonucleotides MRNA092 5 mg/ml 2 ml having 3′-terminal pyrimidine-3′-phosphates. Pyrimidine-3′- monophosphates are also released by RNase A cleavage of adjacent pyrimidine nucleotides. Modified RNA containing pyrimidine-2′-fluoro-dNMPs, such as modified RNA made by in vitro transcription using Epicentre’s DuraScribe T7 Transcription Kits, is completely resistant to cleavage by RNase A. • Remove RNA from DNA preparations • Remove of unhybridized regions of RNA from DNA-RNA or RNA-RNA hybrids

RNase I, E. coli Cat. # Concentration Quantity RNase I degrades ssRNA to nucleoside 3′-monophosphates via RNase I, E. coli 2′, 3′ cyclic monophosphate intermediates by cleaving between N6901K 10 U/µl 1,000 U all dinucleotide pairs. This enzyme is completely inactivated by N6905K 10 U/µl 5,000 U heating at 70°C for 15 minutes, eliminating the requirement to remove the enzyme prior to many subsequent procedures. • Remove RNA from DNA preparations • RNase protection assays to detect single-basepair mismatches in RNA:RNA and RNA:DNA hybrids

RNase III, E. coli Cat. # Concentration Quantity This endoribonuclease specifically digests dsRNA to dsRNA RNase III, E. coli fragments that have 2-base, 3′ overhangs. Complete digestion of dsRNA results in dsRNA fragments of 12-15 bp. RN02950 1 U/µl 50 U • Digest long dsRNA to short dsRNA • RNA structure studies • RNA processing and maturation studies

RNase H, E. coli Cat. # Concentration Quantity This endonuclease specifically degrades the RNA in an RNA:DNA hybrid, without affecting DNA or unhybridized RNA. It will not RNase H, E. coli degrade dsDNA or ssDNA or RNA. E. coli RNase H is inactivated R0601K 10 U/µl 1,000 U at 55°C in 20 minutes. • Eliminate RNA prior to second-strand synthesis of cDNA • Remove poly(A) tails from messenger RNA hybridized to oligo(dT) • Specific cleavage of mRNAs after hybridization to oligonucleotide probes • Specific destruction of “hybrid-arrested” mRNAs during in vitro translation • Diagnostic assays using NASBA™ transcription-based amplification methods • Diagnostic assays based on the Cycling Probe Technology • Template-dependent probe technologies

For Research Use Only. Not for use in diagnostic procedures

18 www.epicentre.com Nucleases, Proteases and Lysozymes

Hybridase™ Thermostable RNase H Cat. # Concentration Quantity Hybridase Thermostable RNase H degrades the RNA in a Hybridase Thermostable RNase H DNA:RNA hybrid, without affecting DNA or unhybridized RNA. H39100 5 U/µl 100 U In contrast to E. coli RNase H, which is rapidly inactivated at H39500 5 U/µl 500 U 55°C, Hybridase RNase H has optimal activity above 65°C, and can be used up to 95°C. This property allows it to be used at temperatures that give the highest hybridization stringency for specific DNA:RNA heteroduplexes, maximizing sensitivity and selectivity while minimizing background due to nonspecific hybridization. • High-stringency hybrid selection • Diagnostic assays of target DNA sequences • Transcription-based amplification methods • High-stringency mapping of mRNA structure

RNase T1, Aspergillus oryzae Cat. # Concentration Quantity This endoribonuclease specifically cuts RNA or deaminated RNA RNase T1, Aspergillus oryzae at the 3′ end of guanosine residues and adjacent nucleotides NT09500K 1,000 U/µl 500,000 U through a 2′, 3′-cyclic phosphate intermediate mechanism. • RNA mapping and structure studies • Remove RNA from DNA preparations • RNA protection assays

™ RiboShredder RNase Blend Cat. # Concentration Quantity RiboShredder Blend is a cocktail of potent RNases that completely RiboShredder RNase Blend degrades unwanted RNA in DNA purification procedures. RS12500 1 U/µl 500 U This highly active cocktail contains a proprietary optimized blend of non-mammalian RNase enzymes. RiboShredder RNase Blend degrades all RNA, converting RNA to nucleoside monophosphates. • Rapidly and completely degrade RNA • DNase-free • Remove RNA from genomic and cloned DNA preparations

RNase R Cat. # Concentration Quantity 3′→ 5′ that digests essentially all linear RNAs but RNase R will not digest lariat or circular RNA structures. Intron RNA can be RNR07250 20 U/µl 250 U isolated from total RNA samples by digestion with RNase R. After digestion, only circular RNAs remain. • Alternative splicing studies • Gene expression studies • CircRNA-Seq sample prep • Intronic screening of cDNA libraries

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 19 Nucleases, Proteases and Lysozymes

Terminator™ 5′-Phosphate-Dependent Cat. # Concentration Quantity Exonuclease* Terminator 5′-Phosphate-Dependent Exonuclease Terminator Exonuclease is a 5′→ 3′, processive exonuclease TER51020 1 U/µl 40 U that degrades RNAs that have a 5′-monophosphate. RNAs with a 5′-triphosphate, 5′-cap structure such as found on most eukaryotic mRNAs or a 5′-OH are resistant to Terminator Figure 1. A 1-hour Terminator Exonuclease reaction. Exonuclease degradation. It will also digest DNA with a 5′-monophosphate. It is not inhibited by proteinaceous RNase A 1-hour Terminator inhibitors. Terminator™ Exonuclease reaction Exonuclease digests the large rRNAs in • Characterize the 5-termini of RNA transcripts a eukaryotic or prokaryotic 1 Hour total RNA sample, • Prepare mRNA-enriched samples from eukaryotic or producing an enriched- prokaryotic total RNA preparations in 1 hour without the use of mRNA preparation. Large Oligo(dT), resins or magnetic beads rRNA mRNA *Patent Pending

OmniCleave™ Endonuclease Cat. # Concentration Quantity This endonuclease digests all forms of DNA and RNA including OmniCleave Endonuclease single-stranded and double-stranded linear, circular, and OC7850K 200 U/µl 50,000 U supercoiled. OmniCleave Endonuclease has the same substrate specificity, and yields the same products as Benzonase®, an enzyme derived from Serratia marcescens. • Improve handling and yield of protein preparations by reducing the viscosity of cell lysates due to nucleic acids • Remove trace contamination by nucleic acids in protein preparations. • Remove host DNA from phage preparations

Proteinase K Cat. # Concentration Quantity Proteinase K digests protein under denaturing conditions such as Proteinase K the presence of SDS, urea or EDTA. The enzyme is often used to MPRK092 50 µg/µl 2 ml remove nucleases during the purification of DNA or RNA. • Purifiy intact, high molecular weight DNA and RNA

For Research Use Only. Not for use in diagnostic procedures

20 www.epicentre.com Nucleases, Proteases and Lysozymes

Ready-Lyse™ Lysozyme Solution Cat. # Quantity Ready-Lyse Lysozyme Solution is a non-mammalian, non-avian, Ready-Lyse Lysozyme Solution recombinant lysozyme preparation for the lysis of Gram-negative R1804M 4 X 106 U (such as E. coli) and Gram-positive (such as Bacillus sp.) bacteria. R1802M 2 X 106 U The specific activity of Ready-Lyse Lysozyme is 200-fold higher R1810M 10 X 106 U than the specific activity of egg white lysozyme. Also, unlike egg Figure 1. Use of Ready-Lyse Lysozyme Solution to white lysozyme, Ready-Lyse Lysozyme Solution is stable at –20°C, recover recombinant proteins. eliminating the need to prepare a fresh solution for each use. The use of Ready-Lyse Lysozyme results in higher yields of protein or nucleic acids than can be obtained with standard egg white M 0 hr. 1 hr. 3 hr. One milliliter of induced cells from lysozyme. a recombinant E. coli clone was • Lyse Gram-negative or Gram-positive bacteria for protein pelleted by microcentrifugation before induction and at 1 and 3 purification or preparations of nucleic acids hours after induction. One milliliter • Isolated from non-mammalian, non-avain source of Ready-Lyse™ Solution was added to each suspension and cells were incubated at room temperature for 30 minutes. The arrow points to the induced protein.

1 2 3 4

EasyLyse™ Bacterial Protein Extraction Solution Cat. # Quantity This extraction solution is designed to lyse bacterial cells to isolate EasyLyse Bacterial Protein Extraction Solution proteins, especially recombinant gene products expressed in RP03750 500 Reactions E. coli, without significant loss of enzymatic activity. It contains a highly active enzyme for cell lysis and a potent nuclease that reduces extract viscosity by digesting all nucleic acids in the sample. The EasyLyse Solution is formulated as a homogeneous reagent for ease of use in high-throughput applications without sonication and gives higher yields of soluble protein. • Rapid protein screening • Easy protein purification • Enzymatic studies • ELISA studies • Manual or robotic procedures

Figure 2. EasyLyse bacterial lysis efficiency. Figure 3. EasyLyse preserves enzyme activity.

EasyLyse™ Bacterial Lysis Efficiency EasyLyse™ Preserves Enzyme Activity

100 Lysis of E. coli aliquots with 1.5 E. coli Lactate Dehydrogenase soluble protein quantities 1.2 80 expressed as a % of total (LDH) specific activity expressed as nmol/min/µg of protein, as determined by the 1.0 60 soluble cell protein. ysis (%) Coomassie Plus Protein Assay.

40 0.5 Bacterial L

20 0.12

0

c170308bnl 0.0 EasyLyse™ Supplier P Specific Activity of Soluble LDH (nmol/min/ µ g) EasyLyse™ Supplier P c180308bnl

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 21 DNA Ligases

T4 DNA Ligase, Cloned Cat. # Concentration Quantity T4 DNA Ligase is a ATP-dependent ligase commonly used T4 DNA Ligase, Cloned for DNA cloning. It covalently joins dsDNA molecules having L0820H 2 U/µl 2,000 U 5′-phosphorylated and 3′-hydroxylated blunt or compatible LH820H 10 U/µl 2,000 U cohesive ends produced by digestion or other For a full line of cloning products, please visit www.epicentre.com enzymatic processes. T4 DNA Ligase has no activity on single- *Please note that this is a Weiss unit. stranded nucleic acids. Following a ligation reaction, T4 DNA Ligase may be inactivated by incubation at 65°C for 10 minutes. • Ligate blunt or cohesive-ended DNA fragments • Repair nicks in double-stranded nucleic acids

E. coli DNA Ligase Cat. # Concentration Quantity This NAD+-dependent enzyme catalyzes the formation of phosphodiester bonds between complementary 3′-hydroxyl and E. coli DNA Ligase 5′-phosphoryl termini of dsDNA. Blunt ends can be ligated in the DL04082H 10 U/µl 200 U presence of condensing reagents such as polyethylene glycol or Ficoll. It is not effective for formation of DNA-RNA or RNA-RNA hybrids. • Molecular cloning of dsDNA with cohesive ends • Blunt-end ligation in presence of 10%-15% PEG and high concentrations of monovalent cations • cDNA cloning of products from second-strand cDNA synthesis experiments

Ampligase™ Thermostable DNA Ligase Cat. # Concentration Quantity Derived from a thermophilic bacterium, Ampligase Thermostable DNA Ligase is stable and active at much higher temperatures than Ampligase DNA Ligase Kit conventional DNA ligases. This enzyme catalyzes NAD-dependent A8101 5 U/µl 1,000 U ligation of adjacent 3′-hydroxylated and 5′-phosphorylated termini A30201 5 U/µl 5,000 U in duplex DNA structures that are stable at high temperatures. Ampligase Enzyme and Buffer Its half-life is 48 hours at 65°C and greater than 1 hour at 95°C. A0102K 100 U/µl 2,500 U It has been shown to be active for at least 500 thermal cycles A32250 5 U/µl 250 U (94°C/80°C) or 16 hours of cycling, which permits extremely A32750 5 U/µl 750 U high hybridization stringency and ligation specificity. No activity is A3202K 5 U/µl 2,500 U detected in ligating blunt-ended DNA, RNA or RNA:DNA hybrids.

• High thermostability allows ligation using high-stringency Ampligase DNA Ligase hybridization conditions A0110K 100 U/µl 10,000 U •  High specificity and stringency permits sensitive detection of A3210K 5 U/µl 10,000 U SNPs One unit of Ampligase DNA Ligase is equal to as many as 15 units of other thermostable DNA ligases. Please compare competitive unit • Ligation Amplification (Ligase Chain Reaction, LCR) definitions. • Repeat Expansion Detection (RED) Ampligase 10X Reaction Buffer • Simultaneous mutagenesis of multiple sites A1905B 10x 5 ml • Other ligation-based detection methods: One unit of Ampligase Ligase is equivalent to at least 15 “cohesive end units” defined by some other major suppliers

For Research Use Only. Not for use in diagnostic procedures

22 www.epicentre.com DNA Ligases

CircLigase™ ssDNA Ligase Cat. # Quantity This thermostable ATP-dependent ligase catalyzes intramolecular CircLigase ssDNA Ligase ligation (i.e., circularization) of ssDNA templates having a CL4111K 1,000 U 5′-phosphate and a 3′-hydroxyl group and ligates ends of ssDNA CL4115K 5,000 U in the absence of a complementary sequence. CircLigase ssDNA Ligase is covered by intellectual property rights licensed to •  Efficient ssDNA ligase activity Epicentre. The purchase of this product conveys to the buyer the non-transferable •  Circularizes ssDNA of >30 bases right to use the purchased product and components of the product in research conducted by the buyer. The buyer cannot sell or otherwise transfer this product or •  Standard reaction conditions produce no detectable single- its components to a third party and in particular, no rights are conveyed to the buyer stranded DNA concatamers or concatameric DNA circles to use the product or its components for commercial use purpose other than for research to gain information that is used by the buyer. • Produce ssDNA templates for rolling circle replication or rolling circle transcription experiments • Produce ssDNA templates for RNA polymerase and RNA polymerase inhibitor assays

Figure 1. CircLigase ssDNA Ligase converts linear ssDNA to circular ssDNA.

1 2 3 4 A 71-base ssDNA oligo was converted to a circular DNA form in a reaction containing – Circular CircLigase™ ssDNA Ligase and ATP. Lane M, DNA markers. Lane 1, 71-base ssDNA. Lane 2, circularization proceeds through an adenylated 70 – 5′ -adenylated intermediate. Lane 3, the closed circular nature intermediate of the reaction product was confirmed by 50 – treating the reaction with exonuclease I, which Linear specifically digests linear DNA. 40 –

CircLigase II ssDNA Ligase Cat. # Quantity This thermostable ligase catalyzes intramolecular ligation (i.e. CircLigase II ssDNA Ligase circularization) of ssDNA templates having a 5′-phosphate and a CL9021K 1,000 U 3′-hydroxyl group and ligates ends of ssDNA in the absence of a CL9025K 5,000 U complementary sequence. • Efficient sDNA ligase activity • Circularize ssDNA of >30 bases • Standard reaction conditions produce no detectable ssDNA concatamers or concatameric DNA circles • Produce ssDNA templates for rolling-circle replication of rolling-circle transcription experiments • Produce ssDNA templates for RNA polymerase and RNA polymerase inhibitor assays

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 23 RNA Ligases

CircLigase RNA Ligase Cat. # Concentration Quantity Thermostable RNA Ligase catalyzes circularization of single- CircLigase RNA Ligase stranded RNA (ssRNA) molecules having a 5′-phosphate and a TRL8101K 100 U/µl 1,000 U 3′-hydroxyl group. • Circularize ssRNA molecules

T4 RNA Ligase 2, Deletion Mutant Cat. # Quantity T4 RNA Ligase 2, Deletion Mutant T4 RNA Ligase 2, Deletion Mutant, T4Rnl2(1-249), is used to ligate single-stranded adenylated DNA or RNA (App-DNA or App- LR2D1132K 2,000 U RNA) oligonucleotides to small RNAs. The preadenylated 5′ ends LR2D11310K 10,000 U of DNA or RNA are ligated to the 3′ ends of RNA. • Prepare cDNA libraries for small-RNA transcriptome analysis such as RNA-Seq • Optimal linker ligation for miRNA cloning

For Research Use Only. Not for use in diagnostic procedures

24 www.epicentre.com Phosphates

APex™ Heat-Labile Alkaline Phosphatase Cat. # Quantity This is an innovative enzyme preparation with improved APex Heat-Labile Alkaline Phosphatase performance over other alkaline phosphatases. APex AP49100 100 Reactions Phosphatase removes the 5′-phosphate from all types of DNA ends, including 5′ protruding, blunt, and 5′ recessed ends, and from RNA ends. The enzyme is irreversibly heat-inactivated by incubation at 70°C for 5 minutes. • Fast, complete and irreversible heat-inactivation for easy transition to next step; no time-consuming substrate purification with phenol:chloroform extraction • Flexible and easy to use—add directly to most RE buffers without supplementation; active over a wide range of temperatures, pH, salts and buffers • Active on blunt, 5′- and 3′-overhang restricted DNA ends for compatibility with any restriction enzyme or experimental design • One simple protocol for most applications • Dephosphorylize DNA vectors prior to cloning to prevent recircularization • Prepare 5′-nucleic acid termini for 5′-end labeling with polynucleotide kinase • Dephosphorylize DNA/RNA substrates for other purposes

RNA 5′ Polyphosphatase Cat. # Concentration Quantity RNA 5′ Polyphosphatase is a Mg2+-independent phosphohydrolase RNA 5′ Polyphosphatase discovered and characterized by Epicentre scientists. The RP8092H 20 U/µl 200 U enzyme sequentially removes the γ and ß phosphates from 5′-triphosphorylated RNA (such as primary RNA transcripts): 5′ pppN—OH 3′→5′ pN—OH 3′ + 2 Pi.

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 25 Kinases, Glucosyltransferases, Deadenylases

T4 Polynucleotide Kinase, Cloned Cat. # Concentration Quantity T4 Polynucleotide Kinase (PNK) catalyzes the transfer of the T4 Polynucleotide Kinase, Cloned (PNK) gamma-phosphate from ATP to the 5′-hydroxyl of ssDNA P0503K 10 U/µl 3,000 U and ddDNA, RNA and nucleoside 3′-monophosphates. The enzyme also removes the 3′-phosphate from 3′-phosphoryl polynucleotides, deoxyribonucleoside 3′-monophosphates, and deoxyribonucleoside 3′, 5′-diphosphates to form a 3′-hydroxyl group. •  Label 5′ termini of DNA and RNA for DNA sequencing, blot- hybridization, or transcript mapping • Phosphorylate of oligonucleotide linkers and other DNA or RNA molecules prior to ligation, or for use in ligation amplification with Ampligase® Thermostable DNA Ligase • Prepare labeled DNA or RNA molecular weight markers for gel electrophoresis and chromatography

T4 Beta-glucosyltransferase Catalog No. Concentration Size T4 Beta-glucosyltransferase is a DNA-modifying enzyme T4 Beta-glucosyltransferase encoded by bacteriophage T4 that catalyzes the transfer of GT11500 10 U/ul 500 U glucose (Glc) from uridine diphosphoglucose (UDP-Glc) to GT112500 10 U/ul 2,500 U 5-hydroxymethylcytosine (5hmC) residues in dsDNA, resulting in the formation of beta-glucosyl-5hmC. • Glucosylate immunodetect 5-hmC DNA • Differentiate 5-hmC from 5 mC • Labele 5-hmC residues by incorporation of a [14C]-UDP- glucose donor into 5-hmC residues

5′ Deadenylase Catalog No. Concentration Size 5′ Deadenylase from yeast (S. cerevisisiae) is a recombinant enzyme that hydrolyzes the 5′-5′ pyrophosphate linkage present 5′ Deadenylase in an activated DNA (Ap-pDNA) or RNA (Ap-pRNA) molecule, DA11101K 10 U/ul 1,000 U releasing adenosine 5′-phosphate and a 5′-phosphorylated nucleic acid. The enzyme has no detectable activity on primary transcripts (5′-monophosphorylated RNA) or capped RNAs. • Deadenylate the 5′ end of DNA or RNA • Aprataxin-dependent DNA repair assays

For Research Use Only. Not for use in diagnostic procedures

26 www.epicentre.com Kinases, Glucosyltransferases, Deadenylases

Uracil N-Glycosylase (UNG) Cat. # Concentration Quantity This Uracil N-Glycosylase (also known as uracil-DNA glycosylase) Uracil N-Glycosylase (UNG) hydrolyzes the N-glycosidic bond between the deoxyribose sugar UG13100 1 U/µl 100 U and uracil in DNA containing deoxyuridine in place of thymidine. UG131K 1 U/µl 1,000 U HK-UNG is active on both ssDNA and ddDNA that contains uracil, but has no activity on RNA or 2′-deoxyuridine-5′-monophosphate. • Repair studies of abasic sites in dsDNA

Uracil-DNA Excision Mix Cat. # Concentration Quantity Uracil-DNA Excision Mix is a blend of enzymes that cleave DNA at Uracil-DNA Excision Mix positions where uracil is present in place of thymine. The Uracil- UEM04100 1 U/µl 100 Reactions DNA Excision Mix is useful for specific or random cleavage of DNA or for DNA repair studies, allowing mapping of uracil residues in any DNA. Uracil-DNA glycosylase in the Excision Mix removes uracil bases from DNA, creating a single-base gap and leaving the deoxyribose phosphate backbone intact. Endonuclease IV in the Excision Mix then cleaves the DNA at each abasic site, leaving a 3′-hydroxyl end and an abasic 5′-phosphorylated end. The minimum oligomer size that will serve as a substrate is 6 base pairs. Uracil-DNA Excision Mix digestion products can be analyzed by denaturing agarose gel electrophoresis or denaturing polyacrylamide gel electrophoresis. • Map uracil-containing residues in any DNA • Map CpG islands • DNA repair studies

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 27 Reverse Transcriptases

MMLV High Performance Reverse Transcriptase Cat. # Concentration Quantity MMLV High Performance Reverse Transcriptase (MMLV HP RT) MMLV High Performance Reverse Transcriptase demonstrates significantly greater reverse transcriptase activity RT80125K 200 U/µl 25,000 U than other commercially available MMLV RT enzymes. Typically, MMLV High Performance Reverse Transcriptase 1st Strand cDNA just 100 units of MMLV HP RT are required for full-length cDNA Synthesis Kit synthesis compared to 200 units of MMLV RT enzymes from MM070150 50 Reactions many other suppliers. • Synthesizes full-length cDNA from RNA templates longer than 15 kb • Efficient cDNA synthesis from picogram amounts of total RNA • Demonstrates about 2X reverse transcriptase activity compared to other commercial MMLV RT enzymes • cDNA synthesis from total RNA or poly(A)-enriched RNA • cDNA synthesis for subsequent PCR or qPCR

Table 1. 3′/5′ ratio analysis of cDNA produced by different reverse transcriptase enzymes.

Epicentre MMLV Competitor I High Performance (RNaseH–mutant of Competitor P Target transcript (size) Reverse Transcriptase MMLV RT) (MMLV RT)

3′/5′ ratio ACTB (1792 b) 0.9 1.7 1.2

3′/5′ ratio GUSB (2162 b) 1.0 6.1 2.5

3′/5′ ratio TFRC (5010 b) 5.5 12.1 11.3

Total cellular RNA from HeLa cells was converted to cDNA using oligo(dT) primer and the three reverse transcriptase enzymes indicated in the Table. A value of 1.0 indicates equal representation of the 3′ and 5′-ends in the cDNA synthesized.

EpiScript™ RNase H– Reverse Transcriptase Cat. # Concentration Quantity EpiScript Reverse Transcriptase is highly efficient at producing EpiScript Reverse Transcriptase full-length cDNA from RNA templates up to 12 kb. This enzyme is ERT12910K 200 U/µl 10,000 U genetically engineered to reduce RNase H activity. This structural ERT12925K 200 U/µl 25,000 U modification eliminates degradation of RNA molecules during first- strand cDNA synthesis and gives EpiScript Reverse Transcriptase superior performance for real-time RT-PCR analysis and other applications. • Efficient cDNA synthesis from picogram amounts of total RNA • Synthesize cDNA for subsequent PCR or qPCR

For Research Use Only. Not for use in diagnostic procedures

28 www.epicentre.com Sample Collection Swabs and Brushes

Sample Collection Swabs and Brushes Cat. # Quantity Epicentre’s swabs and brushes are designed for safe, gentle cell Catch-All Swabs (Hard Pack) collection for subsequent DNA extraction and PCR analysis. Both QEC091H 100 buccal swabs in swabs and brushes are provided individually packaged in sterile hard-pack plastic carrier hard-pack plastic cylinders (hard pack) or as a soft pack. Catch-All Swabs (Soft Pack) QEC89100 100 buccal swabs MasterAmp Brushes (Hard Pack) MB100BR 100 buccal brushes in hard-pack plastic carrier MasterAmp Brushes (Soft Pack) MB100SP 100 buccal brushes

A B

Catch-All Swabs (A) are soft foam swabs on soft, flexible plastic handles. Catch-All Swabs provide gentle, safe sample collection and the porous foam on these swabs catches more of the sample than brushes.

Catch-All Brushes (B) are soft bristle-type sample collection brushes.

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 29 DNA and RNA Extraction

QuickExtract™ Family of Products The QuickExtract DNA Extraction Solutions rapidly and efficiently extract PCR-ready genomic DNA from almost any sample type. The QuickExtract Solutions utilize a simple “heat-and go” process for inexpensive processing of one to hundreds of samples without centrifugation, spin columns or the use of hazardous chemicals. The extracted DNA and RNA should be used for PCR or RT-PCR. Use the MasterPure Purification Kits (page 33) to prepare high purity DNA and RNA for sequencing, cloning, cDNA synthesis, etc.

Products Time Applications Samples Tested

QuickExtract DNA Extraction Solution 3-8 min Genotyping, genetic studies, identity Hair follicles, feathers (quill-end cells), tissue- testing, viral/microbial screening culture cells, buccal cells, zebrafish (organs, (QE0905T, QE09050) scales), mouse tail snips

QuickExtract RNA Extraction Kit 6 min RT-PCR Cultured cells: human, mouse, rat, E. coli, S. aureus (QER09015, QER090150)

QuickExtract Plant DNA 8 min PCR, e.g., GMO testing Arabidopsis, barley, maize, emmer, pepper, Extraction Solution rice, spelt, spinach, soybeans, wheat (QEP80705, QEP70750)

QuickExtract Seed DNA 8 min PCR, e.g., GMO testing Apple, cotton, sunflower, tomato, barley, maize, Extraction Solution oats, rice, rye, wheat (QES08095T, QES080950)

QuickExtract Bacterial DNA 15 min PCR, restriction digests, PFGE, Gram-positive and Gram-negative bacteria Extraction Kit optical mapping (QEB0905T, QEB09050)

QuickExtract FFPE DNA 62 min Microsatellite, single-nucleotide Formalin-fixed, paraffin-embedded human Extraction Kit polymorphisms (SNP), tumor tissue samples heterogeneity studies, copy number (QEF81805, QEF81050) variations (CNV), methylation analysis, short tandem repeats (STR)

QuickExtract FFPE RNA Extraction Kit 32 min RT-PCR Formalin-fixed, paraffin-embedded human tissue samples (QFR82805, QFR82050)

For more information, see: www.epicentre.com/quickextract

For Research Use Only. Not for use in diagnostic procedures

30 www.epicentre.com DNA and RNA Extraction

QuickExtract DNA Extraction Kits Cat. # Quantity QuickExtract products offer a rapid and efficient method for QuickExtract DNA Extraction Solution extracting genomic DNA from virtually any sample for PCR-based QE0905T 5 ml assays. Most samples can be processed in 8 minutes with only QE09050 50 ml two sequential heating steps. • Fast—PCR-ready DNA in 3-8 minutes, ideal for high- throughput workflows • Safe—No organic extraction • Efficient—No columns, transfers, or sample loss

Figure 1. FailSafe PCR amplification of extracted DNA.

M 1 2 3 4 5 6 Buccal cells were extracted using the BuccalAmp DNA Extraction Kit, and all other samples with QuickExtract DNA Extraction Solution. PCR was performed using primers to amplify the regions indicated: Lanes 1-3, human ß-globin (human buccal cells, HeLa cells, and human hair follicle, respectively); lane 4, transgenic mouse GAPDH (mouse tail snip); lane 5, E. coli 16S ribosomal RNA gene (bacteria); lane 6, transgenic SV40 T antigen (mouse tail snip).

QuickExtract FFPE DNA Extraction Kit Cat. # Quantity The QuickExtract FFPE DNA Extraction Kit is a fast, simple, and QuickExtract FFPE DNA Extraction Kit inexpensive method for preparing PCR-ready genomic DNA QEF81805 5 ml from FFPE archival samples. The protocol uses heat treatment QEF81050 50 ml to melt the paraffin, lyse the cells, decrease the formalin-induced cross-linking in the sample, and degrade compounds inhibitory to amplification. • PCR-ready DNA in minutes • No xylene or phenol extraction • No columns, transfers, or sample loss • Extracted DNA is compatible with both real-time and endpoint PCR

Figure 2. PCR amplification of FFPE DNA.

M 1 2 3 4 5 6 7 M DNA was extracted from a slide-mounted, FFPE-preserved human skeletal muscle tissue section with the QuickExtract FFPE DNA Extraction Kit. Two microliters of undiluted, extracted DNA was amplified with primers for three different loci: tumor protein 53 (TP53), dystrophin (DMD), and tumor necrosis factor (TNF). The products were separated on a 3% agarose gel and were visualized with SYBR® Gold. Lane M, 100-bp DNA ladder; lane 1, exon 2 of TP53; lane 2, exon 3 of TP53; lane 3, exon 11 of TP53; lane 4, exon 6 of DMD; lane 5, exon 50 of DMD; lane 6, exon 3 of DMD; lane 7, exon 4 of TNF.

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 31 DNA and RNA Extraction

QuickExtract Plant DNA Extraction Solution Cat. # Quantity The QuickExtract Plant DNA Extraction Solution can be used to QuickExtract Plant DNA Extraction Solution rapidly and efficiently extract PCR-ready genomic DNA from most QEP80705 5 ml plant leaf samples using a simple, one-tube protocol that takes QEP70750 50 ml only 8 minutes. • No bead-beating, freezing, or grinding of plant leaf material • Nontoxic, inexpensive processing • Fast procedure (8 minutes for average sample) • No centrifugation or spin columns to reduce yields • Suitable for high-throughput or automated workflows

QuickExtract Seed DNA Extraction Solution Cat. # Quantity The QuickExtract Seed DNA Extraction Solution can be used to QuickExtract Seed DNA Extraction Solution rapidly and efficiently extract PCR-ready genomic DNA from most QES08095T 5 ml ground seed samples using a simple, one-tube protocol that takes QES080950 50 ml only 8 minutes. • Nontoxic reagents, inexpensive processing • Short procedure • No centrifugation or spin columns • Suitable for high-throughput or automated workflows

QuickExtract Bacterial DNA Extraction Kit Cat. # Quantity The QuickExtract Bacterial DNA Extraction Kit provides a simple QuickExtract Bacterial DNA Extraction Kit method for extracting PCR-ready DNA from Gram-positive and QEB0905T 5ml Gram-negative bacteria. QEB09050 50 ml • Single-tube process • No toxic organic solvents • Suitable for high-throughput applications • Ready-Lyse™ Lysozyme supplied to improve yield

QuickExtract FFPE RNA Extraction Kit Cat. # Quantity The QuickExtract FFPE RNA Extraction Kit provides a fast, simple, QuickExtract FFPE RNA Extraction Kit and inexpensive method for preparing RNA from formalin-fixed, QFR82805 5 ml paraffin-embedded (FFPE) tissue samples for RT-PCR or real-time QFR82050 50 ml RT-PCR. The kit uses heat to melt the paraffin, lyse cells, decrease formalin-induced cross-linking, and degrade compounds that may inhibit amplification. Optional DNase reagents are included for use in some downstream applications. • RT-PCR-ready extracted RNA in 30 minutes, ideal for high-throughput applications • No xylene or phenol extractions • No columns, transfers, or sample loss • Optional protocol allows simultaneous extraction of RNA and DNA from the same tissue sample

For Research Use Only. Not for use in diagnostic procedures

32 www.epicentre.com DNA and RNA Purification

MasterPure™ family of products The MasterPure Kits yield high-purity, high molecular weight DNA and RNA from virtually any sample including FFPE tissue samples. The kits use a safe, gentle, salt-precipitation protocol that eliminates the need for hazardous chemicals and yield-reducing columns. Most protocols can be completed in less than one hour and may be scaled up or down as needed. The DNA and RNA produced can be used for sequencing, cloning, cDNA synthesis and other applications that require high purity, high molecular weight nucleic acid.

MasterPure™ Complete DNA and RNA Cat. # Quantity Purification Kit MasterPure Complete DNA and RNA Purification Kit The MasterPure Complete DNA and RNA Purification Kit enables MC89010 5/10 Purifications efficient purification of intact total nucleic acid (TNA), DNA, or RNA MC85200 100/200 Purifications from every type of biological material. The purified DNA is suitable For isolating TNA, DNA, or RNA. for sequencing and other molecular biology applications. Note: Cat. # MC89010 contains reagents for 10 TNA, 10 DNA, or 5 RNA purifications and Cat. # MC85200 contains reagents for 200 TNA, • Purify DNA in 30 minutes without spin columns 200 DNA, or 100 RNA purifications.

• A260/A280 ratios consistently between 1.8–2.0 • Recover >90% of theoretical DNA yield, including both low- and high-molecular-weight nucleic acids • No phenol, chloroform, or other caustic solvents

Table 1. Typical yields obtained using the MasterPure Complete Kit.

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

*Use the MasterPure™ Yeast DNA Purification Kit to extract yeast DNA.

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 33 DNA and RNA Purification

MasterPure DNA Purification Kit for Cat. # Quantity Blood Version II MasterPure DNA Purification Kit for Blood Version II The MasterPure DNA Purification Kit for Blood Version II can be MB711400 For 400 ml of whole blood used to purify genomic DNA from whole blood or buffy coat. With the optimized protocol for DNA, up to 100 µg of DNA can be Table 1. Typical DNA Yields. recovered from 600 µl of buffy coat in less than 30 minutes. The purified DNA can be used for sequencing, PCR, digestion with Starting Material Yield restriction enzymes, Southern blotting, or other molecular biology 600 µl Buffy Coat 87-109 µg applications. 200 µl Whole Blood 3-9 µg • Purify DNA in 30 minutes without spin columns. 0.6 ml Whole Blood 9-27 µg • A /A ratios consistently between 1.8 and 2.0 260 280 0.8 ml Whole Blood 12-36 µg • Recover >90% of theoretical DNA yield, including both 1 ml Whole Blood 15-45 µg low- and high-molecular-weight nucleic acids 2 ml Whole Blood 30-90 µg • No phenol, chloroform, or other caustic solvents 3 ml Whole Blood 45-120 µg • Scalable protocol 4 ml Whole Blood 60-180 µg 5 ml Whole Blood 75-225 µg

MasterPure Plant Leaf DNA Purification Kit Cat. # Quantity The MasterPure Plant Leaf DNA Purification Kit is designed to MasterPure Plant Leaf DNA Purification Kit purify DNA from 35 to 100 mg of fresh plant-leaf tissue. The MPP92100 100 Purifications purified DNA can be used for sequencing, microsatellite typing, PCR amplification, restriction digestion, Southern blotting, or other molecular biology applications. Plants tested include Arabidopsis, Figure 1. Comparative yields of DNA isolated with the MasterPure Plant Leaf Kit and and a competitor's kit apple, fern, grape, maize, pine, quillwort, sugarcane, sunflower, and tomato. MasterPure Kit Supplier G • Designed for plants high in polyphenolics, like grapevine • Scalable protocol HMW DNA — • Yields high-molecular-weight DNA

1 2 3 4 M 5 6 7 8 M

A 10-µl aliquot of each DNA preparation was separated by electrophoresis on 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.

For Research Use Only. Not for use in diagnostic procedures

34 www.epicentre.com DNA and RNA Purification

MasterPure Yeast DNA Purification Kit • Higher yields of yeast chromosomal DNA than with other kits (Fig. 1) The MasterPure Yeast DNA Purification Kit enables efficient, high- yield purification of high-molecular-weight DNA from yeast and • Recover DNA from a wide variety of yeast species, including other fungi. The protocol involves nonenzymatic cell lysis at 65°C, Candida, Saccharomyces, Pichia, and Schizosaccharomyces, followed by removal of protein by precipitation, and nucleic acid and filamentous fungi such asAspergillus and Penicillium precipitation and resuspension. The protocol can be easily scaled • Purify high-molecular-weight DNA in less than 40 minutes. for larger or smaller samples, including single yeast colonies. The • No bead-beating, columns, or phenol or other organic recovered nucleic acid can be used directly in most applications, extractions including sequencing, PCR amplification, restriction endonuclease digestion, Southern blotting, and genomic library preparation. Cat. # Quantity MasterPure Yeast DNA Purification Kit MPY80200 200 Purifications

Figure 1. Comparative yields of DNA. DNA was quantitated specifically MasterPure Kit – with Hoechst fluorescent dye Supplier Q – C. albicans 33258, which gives minimal Supplier F – fluorescence with RNA. The data represent the average DNA yields MasterPure Kit – S. cerevisiae determined by fluorometry from Supplier Q – two experiments with S. cerevisiae Supplier F – and C. albicans. The MasterPure Kit produced up to 17 times more 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 DNA from C. albicans and 12 times DNA (ng) more DNA from S. cerevisiae than other kits.

MasterPure Gram-Positive DNA Cat. # Quantity Purification Kit MasterPure Gram-Positive DNA Purification Kit The MasterPure Gram-Positive DNA Purification Kit provides all of MGP04100 100 Reactions the reagents needed to purify DNA from Gram-positive bacteria. The purified DNA is suitable for fosmid library construction, PCR, Figure 2. Electrophoretic analysis of DNA purified using restriction digests, Southern blotting, and other molecular biology the MasterPure™ Gram-Positive DNA Purification Kit. applications. The protocol can also be adapted for total RNA purification. M 1 • Scalable method for large sample volumes DNA purified fromB. subtilis • Yields high-quality, high-molecular-weight DNA (ATCC 6051) was separated on a 1% agarose gel and stained with • Lysozyme included; no separate purchase required SYBR® Gold. Lane M, kilobase • Tested on a variety of species ladder; lane 1, 300 ng of B. subtilis DNA. • Can also be used with Gram-negative bacteria 12 kb

1 kb

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 35 DNA and RNA Purification

MasterPure RNA Purification Kit Cat. # Quantity The MasterPure RNA Purification Kit enables efficient purification MasterPure RNA Purification Kit of intact total RNA from a variety of biological materials. The MCR85102 100 Purifications process retains small RNAs that are typically lost when using column-based RNA purification. The purified RNA can be used directly in many molecular biology applications, e.g., RT-PCR, Figure 1. Quality of RNA purified with the MasterPure RNA Northern blots, miRNA analysis, or expression profiling with Purification Kit. microarrays. M 1 2 3 4 5 M • No spin columns, phenol, chloroform, or other caustic solvents • Near-quantitative recovery of even small amounts of RNA — 28 S

Table 1. RNA yields obtained using the MasterPure RNA Purification Kit. — 16 S Sample Sample Size RNA Yield HeLa/HL60 cells 1 x 106 cells 7-15 µg Liver 5 mg 13-25 µg Brain 5 mg 4-11 µg Heart 5 mg 4-5 µg Kidney 5 mg 14-17 µg Thymus 5 mg 9-18 µg Buffy coat 300 µl 3-6 E. coli 3.5 x 106 cells 1.6-1.8 µg Both adherent and suspension cell cultures were purified. A 200-ng aliquot of each RNA preparation was separated by denaturing formaldehyde agarose gel electrophoresis and visualized with SYBR® Gold. Lane M, RNA Marker; lane 1, HeLa cell RNA; lane 2, Ramos cell RNA; lane 3, BDCM cell RNA; lane 4, NIH3T3 cell RNA; lane 5, NRK cell RNA.

Meta-G-Nome™ DNA Isolation Kit Cat. # Quantity The Meta-G-Nome DNA Isolation Kit is used to isolate DNA Meta-G-Nome DNA Isolation Kit from unculturable or difficult-to-culture microbial species present MGN0910 10 Purifications in environmental water or soil samples. The simple extraction procedure involves low-speed centrifugation prior to filtration, and Figure 2. PCR amplification of soil metagenomic DNA. lysis with Ready-Lyse Lysozyme and Proteinase K. The kit isolates high-molecular-weight DNA that is randomly M 1 2 3 4 5 6 sheared (with fragment sizes approximately 40 kb), does not DNA was isolated following require size selection, and can be used directly for end-repair the kit protocol, and PCR was and construction of fosmid libraries. The kit can also be used performed using 16S rRNA primers using various dilutions to prepare 16S rRNA metagenomic libraries for phylogenetic of the template DNA. Lane M, classification or species-abundance studies. Kilobase ladder; lanes 1 and 2, • Isolated DNA is also suitable for PCR (Fig. 2) or sequencing 1 µl and 5 µl of PCR products from 1:10 template dilution; • No bead-beating, agarose plugs, pulse-field gel lanes 3 and 4, 1 µl and 5 µl electrophoresis, or size selection of PCR products from 1:100 • No phenol/chloroform extractions or CTAB template dilution; lane 5, 1 µl of PCR products from undiluted template; lane 6, 1:10 dilution of sample from lane 5.

For Research Use Only. Not for use in diagnostic procedures

36 www.epicentre.com DNA and RNA Purification

MasterPure Yeast RNA Purification Kit Cat. # Quantity The MasterPure Yeast RNA Purification Kit provides all of the MasterPure Yeast RNA Purification Kit reagents needed to purify RNA from yeast cell types (including MPY03100 100 Reactions Candida, Saccharomyces, Schizosaccharomyces, Pichia) and filamentous fungi. The kit uses a rapid salting-out process to remove contaminating macromolecules. The purified RNA is Figure 1. Purity of RNA. suitable for cDNA synthesis and microarray gene expression analysis. • No 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

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.

MasterPure Plant RNA Purification Kit Figure 2. Purification of intact plant RNA from a variety of plant tissues. The MasterPure Plant RNA Purification Kit enables purification of intact total RNA from plant tissues. The RNA obtained is suitable M 1 2 3 4 5 6 7 M for many applications, such as RNA-Seq, RT-PCR, Northern blots, miRNA analysis, and expression profiling with microarrays. Unlike other plant RNA purification kits, a DNase I treatment step is included. Plants tested include banana, citrus, cotton, geranium, grape, maize, maple, raspberry, soybean, strawberry, and tomato leaves, alfalfa sprouts, soybean seedlings and seeds, maize seedlings and mature roots, pine needles, and field mustard seeds. • Optimized technology for plant tissues to remove polyphenols and polysaccharides • No spin columns that reduce yields; small RNAs are purified effectively. • Scalable protocol takes less than 1 hour • No phenol, chloroform, or other caustic solvents Plant tissue RNA was purified using the MasterPure Plant RNA Purification Kit, and 500 ng of each type of RNA was separated on Cat. # Quantity a 1% formaldehyde gel and visualized with SYBR® Gold I. Lanes M, MasterPure Plant RNA Purification Kit RNA ladder; lane 1, maize seedling RNA; lane 2, grape leaf RNA; lane 3, alfalfa sprout RNA; lane 4, strawberry leaf RNA; lane 5, raspberry MPR09100 100 Purifications leaf RNA; lane 6, pine needle RNA; lane 7, maize root RNA.

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 37 PCR

FailSafe™ PCR System • Suitable for multiplex PCR The FailSafe PCR System provides dependable, consistent, • Excellent results with templates up to 85% GC content high-fidelity PCR results for every DNA template, regardless of its • PCR products suitable for both TA cloning and blunt-end source or sequence. First, perform PCR with your template and cloning primer set using all 12 PreMixes provided. For subsequent PCR, • High fidelity PCR simply choose the PreMix that performs the best. Our patented PCR enhancement technology helps with amplifying difficult or Cat. # Quantity GC-rich templates. FailSafe PCR PreMix Selection Kit (all 12 PreMixes)* FS99060 60 Units Figure 1. One-step PCR optimization with the FailSafe PCR PreMix Selection Kit. FailSafe PCR System with PreMix Choice (any one PreMix) FS99100 100 Units The first time... Never Fail . . . FailSafe PCR System with PreMix Choice (any two PreMixes)

™ FailSafe Enzyme Blend WithFS99250 FailSafe™ PCR Systems 250 Units Add your template and primers FailSafe PCR System with PreMix Choice (any eight PreMixes) ™ FailSafe™ 2X PreMixes FailSafeFS9901K Enzyme Blend 1,000 Units Add same template and primers *Choose any FailSafe PCR 2X PreMix (with the above PreMix Choice Kits) FSP995A — FSP995L (PreMixes A-L) 2.5 ml 2X PreMix J FailSafe Enzyme Mix Only ABCD E FGH I J KL FSE51100 100 Units Never Fail . . . FSE5101K 1,000 Units PCR to Select Optimum PreMix J Note: We can only guarantee the failsafe nature of this system if the FailSafe™ Enzyme Blend FailSafe Enzyme Mix is used with a FailSafe PCR 2X PreMix that was first With FailSafe™ PCR Systems Add your template and primers ...and every time determined using the FailSafe PCR PreMix Selection Kit (above).

™ FailSafe PCR Premix Selection Kits: Purchase of this product includes an immunity FailSafe™ 2X PreMixes FailSafe Enzyme Blend from suit under patents specified in the product insert to use only the amount Add same template and primers purchased for the purchaser’s own internal research. No other patent rights (such as 5′ Nuclease Process patent rights) are conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. For complete license statements, see www.epicentre.com/ 2X PreMix J legal.

ABCD E FGH I J KL

PCR to Select Optimum PreMix J

Figure 2. Successful PCR with the FailSafe System. Figure 3. Multiplex PCR of the human CFTR gene.

M 1 2 3 4 5 M A B C D E F G H I J K L

bp 394 ➝

289

Human genomic DNA was amplified using the FailSafe PCR System and primers to a region of the fragile X gene (80%-85% GC-rich). Lanes A-L show the amplification products resulting from PCR using The FailSafe PCR System amplified all five exons of the CFTR gene the 12 FailSafe PCR PreMixes. Lane M, molecular weight marker. In from as little as 1 ng of human genomic DNA. Lane M, DNA marker; this experiment, optimal amplification was obtained with FailSafe PCR lane 1, negative control; lanes 2-5, multiplex PCR from 1, 10, 50, and PreMix J. The location of the expected amplicon is indicated by an 100 ng, respectively, of human genomic DNA. arrow.

For Research Use Only. Not for use in diagnostic procedures

38 www.epicentre.com PCR

MasterAmp Extra-Long PCR Kit Cat. # Concentration Quantity The MasterAmp Extra-Long PCR Kit contains nine MasterAmp MasterAmp Extra-Long PCR Kit Extra-Long PCR 2X PreMixes with dNTPs, buffer, varying amounts MHF9220 50 Reactions of MgCl , and MasterAmp PCR Enhancer with betaine.* Perform 2 MasterAmp Extra-Long DNA Polymerase Mix PCR using all nine PreMixes, one of which will amplify your (Enzyme Mix Only) template. Use this PreMix in all subsequent amplifications. QU92500 2.5 U/µl 500 Units • Fast optimization of long PCR with challenging DNA templates QU9201K 2.5 U/µl 1,000 Units

• Amplify DNA templates up to ~40 kb *Covered by issued and/or pending patents.

MasterAmp Extra-Long PCR Kit; Purchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchaser’s own internal research. No other patent rights (such as 5′ Nuclease Process patent rights) are conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. For complete license statements, see www.epicentre.com/ legal.

Figure 1. Two steps to reliable and consistent PCR results.

Step 1. Find the optimal MasterAmp Extra-Long Step 2. Use the same MasterAmp Extra-Long PCR PreMix to PCR PreMix. obtain reliable and consistent PCR of the same sequence.

M 1 2 3 4 5 6 7 8 9 M 1 2 3 4 5 6 7 8

A. Amplification of a 20-kb region of lambda DNA using MasterAmp Extra-Long PCR 2X PreMixes (1-9). In this example PreMix 4 is optimal. B. Subsequent PCR of a 20-kb lambda DNA region with the same primer pair and MasterAmp PreMix 4 gave consistent results, using the optimal PreMix determined in Step 1.

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 39 PCR

MasterAmp 10X PCR Enhancer with Betaine Cat. # Quantity The MasterAmp 10X PCR Enhancer with betaine* substantially MasterAmp 10X PCR Enhancer with Betaine improves PCR performance, and offers improved sensitivity and ME81205 5 ml specificity for amplification of many DNA templates, including ME81210 10 ml GC-rich templates. Betaine reduces pauses/stops in GC-rich or other difficult regions of the template, thus increasing the yield of PCR products, and reducing the presence of nonspecific PCR products. • Simplify PCR optimization • Enhance PCR yield and specificity • Compatible with most thermostable PCR enzymes *Covered by issued and/or pending patents.

dNTP Solutions Cat. # Concentration Quantity • Sterile, neutral pH solutions of dATP, dCTP, dGTP, dTTP Premixed dNTP Solutions (All 4*) • Available individually or as a PreMix D08104 2.5 mM 10 µmol (4 ml) D59104 25 mM 10 µmol (400 µl) dATP Solution D5910A 100 mM 10 µmol (100 µl) dCTP Solution D5910C 100 mM 10 µmol (100 µl) dGTP Solution D5910G 100 mM 10 µmol (100 µl) dTTP Solution D5910T 100 mM 10 µmol (100 µl) Also available: dUTP Solution D1905U 20 mM 5 µmol (250 µl)

For Research Use Only. Not for use in diagnostic procedures

40 www.epicentre.com In Vitro Transcripton

AmpliScribe™ T7-Flash Transcription Kit Cat. # Quantity The AmpliScribe T7-Flash Transcription Kit uses Epicentre's rapid, AmpliScribe T7-Flash Transcription Kit high-yield in vitro transcription technology. An AmpliScribe ASF3257 25 Reactions T7-Flash Transcription reaction yields more RNA (180 µg per 20-µl ASF3507 50 Reactions reaction) in 30 minutes than other high-yield in vitro transcription kit reactions produce in 2 hours (Fig. 1), including Epicentre's Figure 1. An AmpliScribe T7-Flash transcription reaction own AmpliScribe T7 High Yield Kit. The AmpliScribe T7-Flash is complete in 30 minutes and yields more RNA than other Kit utilizes proprietary enzyme formulations that enable the 2-hour "high-yield" transcription reactions. maximum possible yields of RNA from virtually any template that is downstream of the T7 RNA polymerase promoter, including sequences in linearized plasmids, cDNA, double-stranded oligos, or PCR products. • Exceptionally high yields of full-length RNA. Reactions can be scaled up to obtain grams (or more) of RNA • When transcribing 1 µg of 1-kb linearized plasmid DNA template, a standard AmpliScribe T7-Flash reaction is complete in 30 minutes and yields up to 180 µg of RNA • RNase inhibitor included • Can be used for RNA amplification procedures. However, we recommend TargetAmp 2-Round aRNA Amplification Kits for amplification of mRNA from laser-capture microdissection or other small samples Yields shown were obtained using 1 µg of a linear DNA template in a 20-µl reaction, producing a 1.4-kb RNA.

Poly(A) Polymerase Cat. # Concentration Quantity Adds a poly(A)- tail to the 3′-end of RNA. Poly(A) Polymerase Tailing Kit • Add a poly(A) tail to RNA synthesized in vitro PAP5104H 4 U/µl 50 Reactions • 3′-End-labeling of RNA with radioactive A residues

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 41 In Vitro Transcription

DuraScribe™ T7 Transcription Kit Cat. # Quantity The DuraScribe T7 Transcription Kit produces 2′-fluorine-modified DuraScribe T7 Transcription Kit RNA transcripts—called DuraScript RNA—that are completely DS010910 10 Reactions resistant to RNase A (Fig. 1).1 DuraScript RNA is more stable in DS010925 25 Reactions storage and in all RNA applications, and can be made and used without the tedious procedures normally required for keeping References RNA transcripts intact. Research indicates that double-stranded 1. Capodici, J. et al. (2002) J. Immunol. 169, 5196. DuraScript RNA can be delivered into cultured mammalian cells 2. Meis, J. and Chen, F. (2002) Epicentre Forum 9(1), 10. in the presence of serum and without the need for transfection reagents.2 Double-stranded oligonucleotides, linearized plasmids, or PCR products with a T7 promoter can be transcribed in a DuraScribe transcription reaction. The DuraScribe T7 RNA Polymerase provided in the kit efficiently incorporates 2′-Fluorine-CTP (2′-F-dCTP) and 2′-Fluorine-UTP (2′-F-dUTP), as well as ATP and GTP into full length transcripts. The standard 20-µl DuraScribe reaction, using 1 µg of DNA template, produces approximately 50 µg of DuraScript RNA (Fig. 2). The DuraScribe T7 RNA Polymerase recognizes the same T7 transcription promoter as the standard T7 RNA Polymerase. • DuraScript RNA is resistant to RNase A and DNase • High Yields of DuraScript RNA • T7 in vitro transcription reactions. DuraScribe T7 RNA Polymerase uses the same T7 promoters as the wild type T7 RNA Polymerase • Ideal for RNA aptamer studies. Numerous citations using the DuraScribe Kit with RNA aptamers • SELEX-compatible. DuraScript RNA can be used in SELEX selection processes • Make long or short DuraScript RNA. Double-strand oligos, linearized plasmids, and PCR products with a T7 polymerase promoter can be transcribed

Figure 1. DuraScript RNA is resistant to RNase A digestion. Figure 2. Yield of RNA from a DuraScribe T7 Transcription reaction. M 1 2 3 4

80

60

40

20

DuraScript® RNA Yield (µg) Yield DuraScript® RNA 0 2 hours 4 hours 6 hours Incubation Time

A standard reaction (4-6 hours) produced 40-60 µg of a 1.4-kb DuraScript RNA.

A 1.4-kb standard RNA transcript and a 1.4-kb DuraScript RNA transcript were each incubated with 1 U of highly purified RNase A for 30 minutes. The standard RNA transcript was completely degraded while the DuraScript RNA transcript remained intact. Lane M, size ladder; lane 1, 1.4-kb standard RNA transcript; lane 2, standard RNA after RNase A treatment; lane 3, 1.4-kb DuraScript RNA; lane 4, DuraScript RNA after RNase A treatment.

For Research Use Only. Not for use in diagnostic procedures

42 www.epicentre.com Target Labeling for Expression Microarrays

TargetAmp™ Labeling Kits for Illumina® Cat. # Quantity Expression BeadChip Arrays TargetAmp-Nano Labeling Kit for Illumina Expression BeadChip TargetAmp-Nano Labeling Kit for Illumina Expression BeadChip TAN07924 24 Reactions TAN091096 96 Reactions • Produce microgram amounts of biotin-aRNA from 25 ng to For use with 25-500 ng of RNA. 500 ng of RNA

• Perform the 6-hour TargetAmp-Nano Kit reaction and begin TargetAmp-Pico Labeling Kit for Illumina Expression BeadChip BeadChip hybridization the same day TAP120210 10 Reactions TAP120224 24 Reactions For use with 50-500 pg of RNA TargetAmp-Pico Labeling Kit for Illumina Expression BeadChip • Produce microgram amounts of biotin-aRNA from 50 pg to 500 pg of RNA Figure 1. Genes detected using target labeled with the TargetAmp-Nano and TargetAmp-Pico kits. • Ideal for very small samples, such as laser-capture microdissected (LCM) samples 20000 TargetAmp™-Pico Kit 18000 TargetAmp™-Nano Kit 16000 14000 12000 10000 8000 6000

Number of genes detected 4000 2000

Number of genes detected 0 50 pg 50 pg 100 pg 100 pg 100 ng 100 ng 500 ng 500 ng Liver Muscle Liver Muscle Liver Muscle Ovary Testicle

RNARNA source source and amount amount

Target RNA from the indicated source was labeled using either the TargetAmp-Nano or TargetAmp-Pico kits. The target RNA was hybridized to either the Mouse Ref-8 Expression BeadChip or HumanHT-12 v4 Expression BeadChip (Illumina).

TargetAmp Aminoallyl-aRNA Amplification Kits TargetAmp 2-Round Aminoallyl-aRNA Amplification Kit 1.0 The TargetAmp Aminoallyl-aRNA Amplification Kits generates • Generate microgram amounts of Aminoallyl-aRNA from as little microgram amounts of aminoallyl-labeled antisense RNA as one cell (10 pg of total cellular RNA) (AA-aRNA; also called AA-cRNA) from the total RNA in as little • The entire two-round RNA amplification process can be as one cell. The kits but incorporate a number of improvements completed in 2 days and requires only about 4 hours of that significantly increase the yields of AA-aRNA obtained, while hands-on time reducing the level of nonspecific amplification products. • Highly reproducible amplification and microarray results The aminoally-aRNA produced can be readily labeled using biotin-NHS or fluorescent dye-NHS compounds.

Cat. #. Quantity TargetAmp 1-Round Aminoallyl-aRNA Amplification Kit 101 TargetAmp 1-Round Aminoallyl-aRNA Amplification Kit 101 • Generate microagram amounts of AA-aRNA from as little as TAA1R4924 24 Reactions 25 ng of total cellular RNA TargetAmp 2-Round Aminoallyl-aRNA Amplification Kit 1.0 • The simple, single-tube protocol can be completed in 1 day and TAA2R4924 24 Reactions requires only about 2 hours of hands-on time • Microarray results using AA-aRNA produced with the TargetAmp Kit 101 are highly reproducible

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 43 RNA Amplification

TargetAmp 1-Round aRNA Amplification Kit 103 Cat. # Quantity The TargetAmp 1-Round aRNA Amplification Kit 103 amplifies TargetAmp 1-Round aRNA Amplification Kit 103 the poly(A) RNA in a to generate microgram amounts of amplified TAU1R5124 24 Reactions unlabeled anti-sense-RNA (aRNA; also called cRNA). The kit provides all reagents (except reverse transcriptase and RNA purification columns) for generating unlabeled aRNA. Since each NTP is provided in the kit as a separate solution, researchers can use make labeled aRNA by incorporation of labeled nucleotides during the in vitro transcription step. • Uses 25 ng to 500 ng of total RNA per reaction • Four-hour reaction time • End product is amplified anti-sense mRNA

TargetAmp 2-Round aRNA Amplification Kit 2.0 Cat. # Quantity The TargetAmp 2-Round aRNA Amplification Kit 2.0 amplifies TargetAmp 2-Round aRNA Amplification Kit 2.0 the poly(A) RNA in total RNA. This amplification is sufficient to TAU2R51224 24 Reactions obtain microgram amounts of unlabeled antisense RNA (aRNA; also called cRNA) from a single cell. The kit provides all reagents (except reverse transcriptases and RNA purification columns) for generating unlabeled aRNA. Since each NTP is provided in the kit as a separate solution, researchers can make labeled aRNA by direct incorporation of labeled nucleotides during the finalin vitro transcription step. • Uses 10 pg to 500 pg of total RNA per reaction • Produces microgram amounts of aRNA (cRNA) from total RNA of a single cell • Two-day reaction time and requires only about 4 hours of hands-on time

For Research Use Only. Not for use in diagnostic procedures

44 www.epicentre.com RNA Amplification

MessageBOOSTER™ cDNA Synthesis from Cat. # Quantity Cell Lysates Kit MessageBOOSTER cDNA Synthesis from Cell Lysates Kit A MessageBOOSTER cDNA Synthesis from Cell Lysates Kit MBCL90310 10 Reactions reaction amplifies the poly(A) RNA directly from a cell lysate then converts the amplified RNA to cDNA that is ready for qPCR. There Figure 1. Sensitivity of the MessageBOOSTER Kit. is no need to isolate total RNA. • Low-abundance transcripts in a single cell are readily detected • Hundreds of sensitive qRT-PCRs obtained from as little as a single cell. cDNA can be archived for future use • Linear RNA amplification process preserves the gene expression profile of the cell(s) • No need to purify RNA prior to a kit reaction Relative Fluorescence Units Relative Fluorescence

Cycle

qPCR was performed using undiluted (red), 1:10 diluted (green), 1:100 diluted (blue), and 1:1,000 diluted (purple) cDNA produced from a lysate of a single NRK cell. The low-abundance PBGD transcript was readily detected.

MessageBOOSTER cDNA Synthesis Kit for qPCR Cat. # Quantity The MessageBOOSTER cDNA Synthesis Kit for qPCR enables the MessageBOOSTER cDNA Synthesis Kit for qPCR user to perform sensitive qPCR amplifications using purified the MB060124 24 Reactions total RNA from very small populations of cells, even from as little as one cell The kit amplifies the mRNA (poly(A) RNA) contained in a purified total RNA sample and then converts the amplified RNA to cDNA that is ready for PCR or qPCR. • Amplify mRNA Hundreds of sensitive qRT-PCRs can be obtained from very small amounts of total RNA • Generate large amounts of cDNA from very small samples of intact total RNA for archival purposes • Perform a MessageBOOSTER cDNA Synthesis Kit reaction in 1 day. • The linear RNA amplification and cDNA synthesis processes preserve the gene expression profile.

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 45 Cloning and Competent Cells

™ End-It DNA End-Repair Kit Figure 1. DNA fragments containing any type of ends are rapidly and efficiently converted to5 ′-phosphorylated, blunt- The End-It DNA End-Repair Kit converts DNA containing ended DNA using the End-It DNA End-Repair Kit. 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 5’ next-generation sequencing adaptors, using the Fast-Link 5’ DNA Ligation Kit. The high specific activity of the End-Repair or Enzyme Mix provides complete conversion of protruding ends to 5’ Nebulized, 5′-phosphorylated, blunt-ended DNA. 5’ sonicated, or or sheared DNA The End-It DNA End-Repair Kit contains reagents for 20 or A 50 end-repair reactions (repair of up to 100 µg or 250 µg of 5’ A 5’ genomic DNA, respectively).

End-It™ Blunt-ended, Cat. # Quantity Kit 5’-phosphorylated DNA End-It DNA End-Repair Kit ER0720 20 Reactions 5’ P OH ER81050 50 Reactions OH P5’

The end-repaired DNA is ready for blunt-end ligation using, for example Epicentre’s Fast-Link DNA Ligation Kit.

Fast-Link™ DNA Ligation Kit Cat. # Quantity The Fast-Link DNA Ligation Kit uses a high-quality ligase (Fast- Fast-Link DNA Ligation Kit Link T4 DNA Ligase), to provide extremely rapid, high-efficiency LK0750H 50 Ligations DNA ligation. Cohesive-end ligations can be performed in LK6201H 100 Ligations 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.

Table 1. Fast-Link representative results.

Ligation % White Recombinants Type of End Time Colonies per µg DNA Overhang 5 min 93 2.0 x 106 Blunt 15 min 71 4.4 x 105 PCR product with 1 hr 68 1.2 x 104 3-A overhang

For Research Use Only. Not for use in diagnostic procedures

46 www.epicentre.com Cloning and Competent Cells

CopyControl™ Fosmid Library Production Kit Cat. # Quantity The CopyControl Fosmid Library Production Kits* clone randomly CopyControl Fosmid Library Production Kit sheared DNA to produce unbiased genomic libraries containing 35- CCFOS110 1 Kit to 40-kb inserts. The unique CopyControl technology enables you Complete kit for constructing a CopyControl Fosmid library in the to grow clones at single-copy number to ensure insert stability and Cloning-Ready pCC1FOS Vector. cloning of expressed toxic sequences, and then induce the clones CopyControl HTP Fosmid Library Production Kit to high-copy number (20-50 copies per cell) for high yields of DNA. CCFOS059 1 Kit • Kits include all the components required to construct Complete kit for constructing a CopyControl Fosmid library in the 10 complete libraries. Cloning-Ready pCC2FOS Vector. • Random shearing of genomic DNA ensures more complete and unbiased libraries compared to partial restriction digestion. *Covered by issued and/or pending patents.

Figure 1. Overview of the process for the CopyControl Kits.

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.

TransforMax™ EC100 and EPI300 Competent Cells Cat. # Quantity These highly versatile competent cells provide very high TransforMax EC100 Electrocompetent E. coli transformation efficiency with clones of all sizes. EC10010 10 x 100 µl EC100 Cells—Routine cloning of DNA up to 200 kb. TransforMax EC100 Chemically Competent E. coli Genotype: F– mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 CC02810 10 x 50 µl – ΔlacX74 recA1 endA1 araD139 Δ(ara, leu) 7697 galU galK λ rpsL TransforMax EPI300 Electrocompetent E. coli nupG EC300110 10 x 100 µl EPI300 Cells—Generation of inducible copy-number clones using EC300150 50 x 100 µl the CopyControl Cloning System. TransforMax EPI300 Chemically Competent E. coli – Genotype: F mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 C300C105 10 x 50 µl ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ– rpsL R nupG trfA dhfr. TransforMax EPI300-T1 Electrocompetent E. coli EC02T110 10 x 100 µl • Blue/white screening capability. All selections include pUC19 control DNA. • recA, endA, and restriction minus. • Available in electrocompetent (>1 x 1010 cfu/µg of pUC19) or chemically competent (>5 x 108 cfu/µg of pUC19) preparations. • Choice of regular or T1 phage-resistant strains.

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 47 Cloning and Competent Cells

FosmidMAX™ DNA Purification Kit Cat. # Quantity The FosmidMAX and BACMAX DNA Purification Kits were FosmidMAX DNA Purification Kit developed for easy, reliable isolation of high-quality fosmid or BAC FMAX046 1 Kit DNA for many applications, including sequencing, fingerprinting, Reagents sufficient for 150 x 1.5 ml; 10 x 40 ml; or 5 x 100 ml PCR, and preparation of shotgun libraries. purifications • No columns or binding resins that reduce yields and shear Figure 1. Quality of DNA prepared with the FosmidMAX Kit. the DNA • No toxic organic solvents M 1 2 3 4 5 6 • Very low chromosomal DNA background Fosmid DNA isolated from six different • Ideal for isolating DNA from single-copy clones, and those fosmid clones (lanes 1 to 6) at single- copy number was digested with induced using CopyControl Vectors Hind III and analyzed by agarose gel electrophoresis. Lane M, Kilobase ladder.

Plasmid-Safe™ ATP-Dependent DNase Cat. # Concentration Quantity Plasmid-Safe ATP-Dependent DNase selectively removes Plasmid-Safe ATP-Dependent DNase contaminating bacterial chromosomal DNA from cosmid, BAC, E3101K 10 U/µl 1,000 U fosmid, and plasmid preparations. The enzyme will processively E3105K 10 U/µl 5,000 U degrade linear DNA from the ends; closed circular DNA (i.e. a E3110K 10 U/µl 10,000 U plasmid) does not have free ends, and is therefore not degraded.

These properties make Plasmid-Safe ATP-Dependent DNase Figure 2. Plasmid-Safe ATP-Dependent DNase removes ideal for BAC and fosmid purification protocols such as shotgun contaminating genomic DNA from plasmid preps. sequencing and FISH where high purity DNA is necessary. • Remove contaminating bacterial chromosomal DNA in large- M – + scale plasmid, cosmid, fosmid, BAC or vector preparations –, mixture of 3 µg of digested bacterial chromosomal DNA and 500 ng of uncut plasmid before Plasmid-Safe DNase treatment +, mixture of chromosomal DNA and plasmid DNA after Plasmid-Safe™ DNase treatment (incubated with Plasmid-Safe DNase for 30 minutes at 37°C); M, kb ladder.

For Research Use Only. Not for use in diagnostic procedures

48 www.epicentre.com Reverse Forward Forward Sequencing Sequencing Reverse PCR Primer Primer Primer PCR Primer pMOD™-2 Pvu II pUC-19 Multiple Pvu II Vector ME EcoRI HindIII ME R PshAI Cloning Site PshAI (colE1 ori, Amp )

Reverse Forward Forward Sequencing Sequencing Reverse PCR Primer Primer Primer PCR Primer R6Kγ ori pMOD™-3 Pvu II pUC-19 Multiple Pvu II Vector ME EcoRI HindIII ME R PshAI Cloning Site PshAI (colE1 ori, Amp )

In Vitro Use

Target DNA Transposon Transposase

1. Incubate 37°C; 2 hrs 2. Transform E. coli 3. Select Drugr clones

4. Prepare template DNA

Bacterial Mutagenesis

Introduction to Bacteria Mutagenesis using EZ-Tn5™ Transposomes and In Vivo Transposomics

An EZ-Tn5 Transposome provides a rapid and easy method for Figure 1. An EZ-Tn5 Transposome is the stable complex generating a library of random gene knockouts in living bacteria. formed by EZ-Tn5 Transposon with EZ-Tn5 Transposase in 2+ An EZ-Tn5 Transposome is a stable complex formed between the absence of Mg . an EZ-Tn5 Transposon (a modified Tn5 transposon) and EZ-Tn5 Transposase (a hyperactive Tn5 Transposase) in the absence of In Vivo Use Mg 2+ (Figure 1). ME ME

EZ-Tn5 Transposomes are so stable that they can be introduced minus Mg++ into living bacteria that can be transformed by electroporation. 2+ EZ-Tn5™ 10 min, 37°C Once in the cell, the EZ-Tn5 Transposase is activated by Mg Transposon in the host cell and the EZ-Tn5 Transposon DNA is randomly EZ-Tn5™ inserted into the host's genomic DNA. Since there is no need for Transposome™ cell conjugation, suicide vectors, or specific host factors, EZ-Tn5 Transposomes are ideal for quickly and easily generating libraries Phenotypic Analysis of random gene knockouts, developing novel strains of bacteria or Insertion Clones "tagging" bacteria for special applications. 1. Electroporate Direct Genomic Figure 2.2. An Plate EZ-Tn5 Transposome™ Complex. DNA Sequencing

Cells Selection Plate What EZ-Tn5 Transposomes are available and how EZ-Tn5™ are they used? Transposome™ Rescue Cloning

EZ-Tn5 Transposomes are available with either a kanamycin- Insertion Clones resistance (Kanr) marker or a dihydrofolate reductase gene (DHFR; 1. Electroporate trimethoprim selection) marker. 2. Plate Simply electroporate the EZ-Tn5 Transposome of choice into the Selection Plate electrocompetent bacteria of choice and allow the cells to recover. EZ-Tn5™ Cells Transformed bacteria are selected on the appropriate medium Transposome containing kanamycin or trimethoprim. Surviving colonies contain an EZ-Tn5 Transposon randomly inserted into their genomic DNA. The number of transposition clones obtained is dependent on the transformation efficiency of the host cell. Genomic DNA The EZ-Tn5 Transposome system has been optimized so that on average just one EZ-Tn5 Transposon is inserted in the DNA of a cell. Transposon-insertion clones can be screened (Figure 2) Phenotype Sequencing using a phenotypic assay defined by the user, by genomic DNA Screening sequencing, or PCR. An EZ-Tn5 Transposome Complex can be electroporated into living A Broad Host Range System cells where it randomly inserts the transposon component into the host's genomic DNA. The EZ-Tn5 Transposon insertion site can be Researchers have been using EZ-Tn5 Transposomes for more analyzed by a variety of methods. than 15 years with more than 60 different species reported in the literature. Both Gram-negative and Gram-positive bacteria have been successfully transformed. The most important requirement for success is preparation of electro-competent bacteria with the highest transformation efficiency possible.

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 49 Bacteria Mutagenesis

EZ-Tn5 Tnp and EZ-Tn5 Cat. # Quantity Tnp Transposome Kits EZ-Tn5 Tnp Transposome™ Kit EZ-Tn5 Transposome complexes are formed between an EZ-Tn5 TSM99K2 10 Reactions Transposon and EZ-Tn5 Transposase, and provide a simple and EZ-Tn5 Tnp Transposome Kit reliable method for generating a library of random gene knockouts TSM99D1 10 Reactions 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 Figure 1. Overview of in vivo transposition. 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 Insertion Clones the EZ-Tn5 Transposon (Fig. 1). 1. Electroporate • Rapid mutagenesis procedure is simpler and easier to use than 2. Plate chemical mutagenesis. Selection Plate • More efficient than using mini-transposons with suicide EZ-Tn5™ Cells plasmids. Transposome • 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 EZ-Tn5 Transposome complexes, is covered by U.S. Patent The EZ-Tn5 Transposon insertionGenomic site DNA in bacterial DNA can be No. 6,159,736 and related patents and patent applications, exclusively licensed to sequenced directly using genomic DNA isolated using the MasterPure Epicentre. See www.epicentre.com/legal. Complete DNA Purification Kit and primers homologous to the ends of the transposon. Phenotype Sequencing Screening

EZ-Tn5 Tnp Transposome Kit Cat. # Quantity Nothing makes the cloning process easier than creating mutations EZ-Tn5 Tnp Transposome Kit in vivo with the EZ-Tn5 Tnp Transposome Kit.* TSM08KR 10 Reactions In addition to encoding a broad host-range kanamycin-resistance gene, the transposon contains an E. coli conditional origin of *The use of Transposome complexes for in vivo insertion of a transposon, including, replication (R6Kγori). The presence of this origin of replication but not limited, to EZ-Tn5 Transposome™ complexes, is covered by U.S. Patent No. 6,159,736 and related patents and patent applications, exclusively licensed to enables the propagation or “rescue” of the region of genomic Epicentre. See www.epicentre.com/legal. DNA, or plasmid, into which the transposon has been inserted (Fig. 2). • 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 . • Rescue clones can be sequenced bidirectionally using the primers provided that are homologous to the ends of the transposon.

Figure 2. Overview of rescue cloning of transposon insertion sites.

ori KanR ori ori ori ori KanR KanR KanR R Transform pir E. coli Kan ori ori KanR and select on Kan plates KanR KanR rescued clones Rescued plasmid DNA Purify, then shear or clone Self-ligation digest genomic DNA

For Research Use Only. Not for use in diagnostic procedures

50 www.epicentre.com Bacterial Mutagenesis

EZ-Tn5 , EZ-Tn5 , and Figure 1. The process for generating DNA sequencing templates using an EZ-Tn5 Insertion Kit. EZ-Tn5 Insertion Kits EZ-Tn5 Insertion Kits* are designed to simplify and speed up complete sequencing of any cloned DNA >2 kb, without primer Target walking or subcloning. A simple, one-step in vitro reaction results DNA in random insertion of a single EZ-Tn5 Transposon containing a EZ-Tn5™ Transposon EZ-Tn5™ Transposase selectable marker into the clone. Then, transform E. coli cells with an aliquot of the reaction and select for the marker encoded by 1. Incubate at 37°C, 2 hr the EZ-Tn5 Transposon. Use the primers provided in the kits to 2. Transform E. coli sequence insertion clones bidirectionally from primer binding sites 3. Select Drugr clones 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. • Use a single set of sequencing primers (provided in the kits) to completely sequence any clone.

*Covered by intellectual property rights licensed to Epicentre. Sequence bidirectionally from primer-binding sites Cat. # Quantity EZ-Tn5 Insertion Kit EZI982K 10 Reactions EZ-Tn5 Insertion Kit EZI921T 10 Reactions Select inserts on kanamycin, tetracycline, or trimethoprim plates. EZ-Tn5 Insertion Kit EZI912D 10 Reactions

Cat. # Quantity EZ-Tn5 Insertion Kit EZ-Tn5 Insertion Kit The EZ-Tn5 Insertion Kit* facilitates the sequencing and genetic analysis of plasmids or any other EZI011RK 10 Reactions circularized DNA that would not otherwise replicate in E. coli. The kit is based upon the EZ-Tn5 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.

EZ-Tn5 Custom Transposome Construction Kits Cat. # Quantity The new EZ-Tn5 Custom Transposome Construction Kits EZ-Tn5 Custom Transposome Construction Kit with pMOD-2 Vector provide the key reagents needed for constructing your own Transposomes. Also included are the standard pMOD PCR TNP10622 20 reactions primers, 10X EZ-Tn5 Reaction Buffer for in vitro reactions, detailed EZ-Tn5 Custom Transposome Construction Kit with protocols for creating and purifying the custom transposons, pMOD-3 Vector and stop solution for in vitro reactions. Sufficient reagents are TNP10623 20 reactions provided to create Transposomes for up to 10 in vitro or 20 in vivo transposition reactions.

For Research Use Only. Not for use in diagnostic procedures

(800) 284-8474 51 Molecular Biology Solutions. Epicentre Enzymes and Reagents for CRISPR/Cas9 Genome Editing sgRNA in vitro transcription AmpliScribe T7-Flash Transcription Kit (p. 41) – Transcribe more RNA in 30 minutes than other kits produce in 2 hours DuraScribe T7 Transcription Kit (p. 42) – Transcribe RNA that is completely resistant to RNase A sgRNA Cloning Fast-Link DNA Ligation Kit (p. 46) – Rapid, high-efficiency ligation of blunt and cohesive end DNA TransforMax Competent Cells (p. 47) – high transformation efficiency electrocompetent and chemically competent E. coli Plasmid-Safe DNase (p. 17) – Digest residual linear DNA for improved sgRNA cloning

Analysis of CRISPR/Cas9 mutated DNA QuickExtract DNA Extraction Solution (p. 31) – Extract PCR-ready DNA in 8 minutes from CRISPR/Cas9-mutated cells QuickExtract Plant DNA Extraction Solution (p. 32) – Rapid extraction of PCR-ready DNA from CRISPR/Cas9-mutated plant cells

CRISPR/Cas9 genome editing complex

Phone: 800-284-8474 I 608-258-3080 Fax: 608-258-3089 www.epicentre.com