Amplify

Purify

Analyze PCR Reagents PCR Reagents Everything you’d expect for PCR*.USB Taq and new Taq Master Mixes. Three new kits for RT-PCR. Nucleotides and binding proteins. Tested User Friendly™ (TUF) . And ExoSAP-IT®, the revolutionary clean-up tool that eliminates dNTPs and primers fast, with no columns and no sample loss. At USB, we have everything you really need for PCR. We test and re-test all our reagents to ensure they work reliably the first time, every time. We know you’d expect nothing less.

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2 Amplify

Purify

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3 4 Table of Contents SEQUENCING KITS ANALYZE PURIFY AMPLIFY M ees rncits AVR)27 27 24 23 24 M-MLV ReverseTranscriptase (M-MLV-RT) AMV ReverseTranscriptase (AMV-RT) T7 Gene6Exonuclease Lambda Exonuclease I Shrimp (DNase) Calf IntestinalAlkalinePhosphatase(CIAP) Shrimp AlkalinePhosphatase(SAP) A-Taq-IT hroSqeaeCceSqecn i 28 28 28 28 SequenaseCycle SequencingKit Thermo SequenaseRadiolabeled TerminatorThermo CycleSequencing Kit SequenaseDyePrimerManualCycleSequencing Kit Thermo Sequenase PCRProduct SequencingKit ExoSAP-IT T rclDAGyoyae etLbl 18 16 Uracil-DNA Glycosylase,Heat-Labile Uracil-DNA Glycosylase T4 Gene32Protein Single-Stranded DNABindingProtein (SSB) RT T One-Step RT-PCR Kit PCR Nucleotides T T T β Product aq PCRMasterMix aq PCRKit aq DNAPolymerase aq PCRMasterMixPlus wo-Step RT-PCR Kit -Agarase I

Script Kit Phone: 1-800-321-9322 | Fax: 1-800-535-0898 ™ ® NEW NEW NEW NEW NEW NEW Page 26 25 25 23 22 20 12 18 17 10 14 6 9 8 8 7 Table of Contents 5 29 29 29 29 29 29 19 29 19 29 29 29 30 19 Page www.usbweb.com eaction buffer and/or dilution buffer. eaction buffer TUFª User Friendly): (Tested This symbol used on USB modifying enzymes indicates an enhanced level of qualification, including functional testing for specific applications and/or pertinent as the testing, as well TUF Typically, activity assay. standard modifying enzymes include a qualified r ater, Nuclease-Free ater, ater, Nuclease-Free, Endotoxin-Tested Nuclease-Free, ater, PCR Qualified ater, 30 ater, RNase-Free, DEPC-Treated RNase-Free, ater, 30 ris/HCl, 1M Solution, pH 7.0ris/HCl, 1M Solution, pH 7.5ris/HCl, 1M Solution, pH 8.0 30 30 30 Product Acridine Orange Agarose, Low Melting/Gelling Temperature Melting/Gelling Low Agarose, Agarose mg/ml SolutionAlbumin, Bovine, 50 0.5M Solution EDTA, Ethidium Bromide Tablets Ethidium Bromide Formamide Gelatin Glycerol Magnesium Chloride, 25mM SolutionMineral Oil, Light Phenol, pH 4.5, EquilibratedPhenol, pH 8.0, EquilibratedPhenol: Chloroform: Isoamyl Alcohol (25:24:1)3M Potassium Acetate Solution, pH 5.52M Potassium Chloride Solution RecombinantRNase Inhibitor, 3M Sodium Acetate Solution, pH 5.5 29 5X Solution TBE Buffer, Solution 1X TE Buffer, Solution 50X TE Buffer, T 19 T T W 19 29 29 29 29 29 29 29 W W W PCR RELATED REAGENTS PCR RELATED 6 Amplify Master Mix T aq PCR NEW Phone: 1-800-321-9322 | Fax: 1-800-535-0898 Master Mixtoprimers,DNAtemplateandPCR-QualifiedH several pipettingsteps.Fora50µlreaction, simplyadd25µlofTaq PCR The mixsavestimeandreduces potentialcontaminationerrors byeliminating Convenient thoroughly QCtested,experimentalvariabilityissignificantlyreduced. amplification. Sincethemixisprepared from highqualityreagents andis PCR MasterMixprovides inPCR forrobust andreliable performance r containingUSBTaqformulation DNAPolymerase,Ultrapure nucleotidesand USB Taq PCRMasterMixissuppliedasaconvenient2Xpre-mixed T Store at-20°C.Mixwellpriortouse. Shipping andStorage: 20mM Tris-HCl (pH8.6),100mMKCl,3mM MgCl T lambda DNAsequence. PCR protocol. Thisprotocol isbasedupontheamplificationofa500bp Functionally testedforPCRaccording tothestandard Tested UserFriendly Functional Test: pages 14-15).Qualityresults are alsoobtainedwithlongPCRproducts (Fig.4). targets aswelllowtomoderatelyabundantcDNAs(Figs.1-2;Figs.1-3, The mixmaybeusedtoamplifyPCRproducts from low-copygenomicDNA Sensitive andRobust bycolonyPCR(Fig.3)ortransgenicstudies. screening transformants USB Taq PCRMasterMixisidealforhigh-throughput applicationssuchas Ideal forHighThroughput The end-usermayset-up10µl,2550µlor100reactions. Scaleable stable atroom temperature (Fig.2). fact, rigorous stabilitystudieshavebeendoneatUSBshowingthemixtobe (Fig.1).Itisalsostableat4°Cforextendedperiodsoftime.In in performance The mixwithstandsrepeated freeze-thaw cycleswithnoobserveddecrease Stable MgCl dCTP, dGTP, dTTP),50 units/mlTaq DNAPolymerase,stabilizers.Additional eaction buffer optimizedforawidevarietyofPCRapplications.UseTaq aq PCRMasterMix aq PCRMasterMixFormulation(2X): effect on performance isobserved. effect onperformance target copies.Following25freeze-thaw cycles,nosignificant 50 pghumangenomicDNA.Thisrepresents about30total Amplification ofasegmentthesingle-copyNumbgenefrom Freeze-Thaw StabilityofUSBTaq PCRMasterMix. 0.4 kbÐ 1.0 kbÐ 2 may beaddedforoptimizingPCRconditions. M neg ctrl Figure 1 Freeze 01525 Thaws ← (455 bp) Numb 2 , 0.4mMdNTPs(dATP, (247 bp) p53 storage conditions,PCRamplificationisnotsignificantlyaffected. genomic DNA.(Thisrepresents about30totaltargetcopies.)Underthese p53 gene(left)andNumb(right)were amplifiedfrom 50pghuman was compared againstafreshly prepared mix.Segmentsofthesingle-copy stored at4°Candroom temperature fortwomonthsanditsperformance 4¡C andRoomTemperature Stability. 0.5 kbÐ 0.1 kbÐ 2 O. → M neg ctrl Fresh 4¡CRT Figure 2 0X1m $32.00 10 X1ml 71785 Ultrapure PCR-Qualified Water, $76.00 (50 100 reactions 71162 Card. 625 µlwithaBriefProtocol supplied inapackas4x T T aq PCRMasterMixis aq PCRMasterMix µ l/rctn) USB Taq PCRMasterMixwas M neg ctrl Fresh 4¡CRT ← (455 bp) Numb Amplify 7 Numb (4.6 kb) ← Segments of the single- l/rctn) M µ aq PCR Master Mix Plus T 71163 100 reactions(50 $89.00 3.0 kb Ð 5.0 kb Ð Figure 4 Figure ab M → 1.0 kb Ð 2.0 kb Ð Amplification of Long Targets from Genomic DNA. Amplification of Long Targets 50 pg and amplified from Numb gene (b) were copy NRAGE gene (a) and Mix. PCR Master USB Taq using 50 ng of human genomic DNA respectively NRAGE (1369 bp) for added convenience. 2 O and MgCl Colony PCR (574 bp) 2 www.usbweb.com ← 2 Order online at www.usbweb.com. M5248 colonies using USB Taq M5248 colonies using USB E. coli Figure 3 q PCR Master Mix Plus is supplied as a kit with the following components: q PCR Master Mix Plus is supplied as a aq PCR Master Mix Plus aq PCR Master Mix Plus includes the USB Taq PCR Master Mix plus PCR Master Mix Taq aq PCR Master Mix Plus includes the USB Brief Protocol T T PCR-Qualified H Ultrapure 4 x 625 µl Taq PCR Master Mix, 2X 4 x 625 µl Taq 3 x 1 ml PCR-Qualified Water 1 x 1 ml 25mM MgCl Functional Test User Friendly Tested to the standard Functionally tested for PCR according amplification of a 500 bp is based upon the This protocol PCR protocol. lambda DNA sequence. Ta ctrl neg M Amplification of a segment of the T4 DNA Polymerase gene Amplification of a segment NEW aq PCR 1.0 kb Ð 0.5 kb Ð Master Mix Plus T Colony PCR. in several different overexpressed PCR Master Mix. 8 Amplify Polymerase Taq Ta NEW q PCRKit DNA Phone: 1-800-321-9322 | Fax: 1-800-535-0898 100mM Tris-HCl (pH8.6),500mMKCl,15mMMgCl (1 mlincluded,PN71165): Functionally Tested 10XPCRReactionBuffer T T 100mM KCI,50%glycerol, stabilizers. 20mM Tris-HCl, pH8.5,1mMDTT, 0.1mMEDTA, Storage Buffer: 5 units/µl Concentration: .Innis,M.A.,Myambo, K.B.,Gelfand,D.H.,andBrow, 1. References: Colony PCRfordetectionofclonedinserts. 2. Amplification ofDNA. 1. Applications: 25mM solution. .Innis,M.A.andGefland,D. H.(1990) 3. Lawyer, F. C.,Stoffel, S.,Saiki,R.K.,Myambo, 2. Functionally Tested MgCl Taq Taq yield andlengthinPCRamplification aquaticus 25mM MgCl T significant decrease inenzymeactivity, andissuitableforroutine PCR.USB DNA Polymerasewithstandsrepeated incubationsat95°Cwithouta has nodetectablecontaminatingendonucleaseorexonucleaseactivities.Taq activity anda5’–›3’ exonucleaseactivity. USBTaq isfunctionallytestedand Polymerase. Thisenzymepossessesahighlyprocessive 5’–›3’ polymerase 10X PCRBuffer withoutMgCl 10X PCRBuffer 10mM PCRNucleotideMix,Ultrapure T Kit Components: 25mM magnesiumchlorideforoptimizingPCRconditions. standard reactions, onewithandwithoutMgCl PCR toensure reliable results. ThekitalsoincludestwoPCRbuffers for Wa wide varietyoftemplates.Allcomponents,includingUltrapure PCRQualified The USBTaq reagents toamplifyDNAfrom PCRKitincludesthenecessary a T PCR QualifiedWater, Ultrapure 25mM MgCl ested inastandard PCR. aq DNAPolymeraseissuppliedwith10XPCRBuffer plusaseparatetubeof aq DNAPolymerase,250units se srFinlªFunctionalTest: ested UserFriendlyª aq PCRKit M. A.(1988) A GuidetoMethodsandApplications 264 Drummond, R.andGelfand,D.H.(1989) ter andUltrapure PCRNucleotideMix,are functionallytestedinstandard †† N Polymerase DNA DNA Polymerase is a thermostable enzymederivedfrom DNA Polymeraseisathermostable , 6427-6437. which ispurifiedfrom 2 2 for optimizingPCRconditions.Eachlotistestedproduct Proc. Natl.Acad. Sci. 2 , (1mlincluded,PN71167): 2 E. coli (1) 85 . overexpressing nativeTaq DNA , 9436-9440. , AcademicPress. PCR Protocols: J. Biol.Chem. 2 2 , andaseparatetubeof . Thermus from fragmentsrangingfrom1kbto6amplified DNA 0.5 kb λ 12 kb 1 kb 2 kb 5 kb DNA. l$19.00 1 ml 71165 T Custom SizesByRequest $1,520.00 5,000 units $485.00 $390.00 $104.00 $28.00 5 x250units 1,000 units 250 units 50 units 71160 Taq (100 $152.00 100 reactions 71161 T aq ReactionBuffer, 10X aq PCRKit Figure 1 N Polymerase DNA µ l/rctn) Amplify 9 $58.00 $58.00 $145.00 l l l µ µ µ mol (5 ml) $743.00 mol (1 ml)mol (5 ml) $176.00 $743.00 mol (1 ml)mol (5 ml) $176.00 $743.00 mol (1 ml) mol (5 ml) mol (1 ml)mol (5 ml) $176.00 $743.00 mol (1 ml)mol (5 ml) $176.00 $743.00 mol (1 ml)mol (5 ml) $176.00 $743.00 mol (1 ml) $176.00 µ µ µ µ µ µ µ µ µ µ µ µ µ µ mol (0.25 ml) µ mol (0.250 ml) $55.00 mol (0.250 ml) $55.00 mol (0.250 ml) $55.00 mol (0.250 ml) $55.00 mol (0.250 ml) $55.00 mol (0.250 ml) $55.00 µ µ µ µ µ µ 4 dNTPs per pack (pk) $564.00 4 dNTPs per pack (pk) $2,275.00 100mM 25 100mM 25 100mM 25 100mM 25 100mM 25 100mM 4 x 25 100mM 25 www.usbweb.com Order online at www.usbweb.com. onvenient packaging – dNTPs are available separately, as a set of four or as a PCR mix. as a set of four available separately, are packaging – dNTPs onvenient ll Ultrapure dNTPs are functionally tested in long PCR to generate a 20.7 kb fragment. to generate a 20.7 tested in long PCR functionally are dNTPs ll Ultrapure •A •C ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE 10mM each dATP, dCTP, dGTP, dUTP dGTP, dCTP, 10mM each dATP, 10mM 500 Ultrapure dTTP dGTP, dCTP, 25mM each dATP, PCR Nucleotide Mix with dUTP Ultrapure 25mM 500 dUTP (2'-Deoxyuridine-5'-Triphosphate)UltrapurePCR Nucleotide Mix Ultrapure dTTP dGTP, dCTP, 10mM each dATP, PCR Nucleotide Mix 10mM 100 500 500 UltrapuredTTP (2'-Deoxythymidine-5'-Triphosphate)Ultrapure 100 500 500 dGTP (2'-Deoxyguanosine-5'-Triphosphate)UltrapuredITP (2'-Deoxyinosine-5'-Triphosphate) 100 100 500 (2'-Deoxyadenosine-5'-Triphosphate)UltrapuredCTP (2'-Deoxycytidine-5'-Triphosphate)Ultrapure 100 100 500 500 dATP, dCTP, dGTP, dTTP dGTP, (Set of Four) dCTP, dATP, (2'-Deoxynucleoside-5'-Triphosphates)dATP 4 dNTPs per pack (pk) $188.00 77330 77119 77212 77206 77108 77205 77106 77104 77102 77128 100mM 4 x 500 77100 77328 100mM 4 x 100 Prod. No. Product Description [conc] Size Price USB ULTRAPURE Nucleotides 10 Amplify RT-PCR Kit One-Step NEW Phone: 1-800-321-9322 | Fax: 1-800-535-0898 M-MLV ReverseTranscriptase diverse RNAsamplesandcustomprimersprepared bystandard approaches. Thekitisbasedon The USBOne-StepRT-PCR Kitcomescompleteandready touseforRT-PCR. Thekitcanbeusedwith Complete One-StepRT-PCR Kit simplicity andconvenienceminimizesthepossibilityforcontamination. r cDNA toyielddouble-strandedproduct. OnestepRT-PCR isavariationonRT-PCR inwhichall DNApolymeraseamplifiesthesingle-stranded single-stranded cDNA.InthePCRstep,athermostable (cDNA) product DNA amplifying asingle-strandedRNAtemplatetoyieldabundantdouble-strandedcomplementary Reverse transcription-polymerasechainreaction, orRT-PCR, and isamethodforconverting •A •S • • format. outRT-PCRDesigned forsimplicityandconvenienceincarrying inaone-tube One-Step RT-PCR Kit supplements foramplifyingG+Crichtargets,enablespecificamplificationothers(Fig.3). targets. Afewsimpleprotocol adjustments,suchasoptimizationoftheamountprimersoraddition amounts ofRNA(Fig.2).Standard reaction conditionsare sufficient forspecificamplificationofmost 1 µgtotalRNAor100pgtongpolyARNA.Highlyexpressed targetscanbedetectedinevenlower PCR products (Fig.1).Moderatelyorweaklyexpressed targetscangenerallybedetectedin1ngto (~0.2to1.5kb) The kitcanbeusedfordetectionofdiverseRNAtargetsbasedongenerationshort Highly Sensitive,Specific re dNTPs, supplementalmagnesiumchloride,andRNase-free waterare alsoincluded,allowingquick, optimized tobalancesensitivityandspecificity. Anoptimizedreaction buffer, RNaseinhibitor, Ultrapure eaction components are mixed in one tube prior to starting thereactionseaction componentsare mixedinonetubepriortostarting liable setupofreactions. can beamplifiedsuccessfullywithoutneedforoptimization Analysis ofspecificRNAsplicevariants samples Qualitative analysisofexpression ofoneorafewgenesinmultipleRNA treamlining theoptimizationofRT andPCRstepssimultaneously starting pointforanalysisofnewRNAtargets,giventhatmanytargets starting (1,2) . IntheRT step,theRT enzymereverse transcribesanRNAtemplate,yielding were sizes. designedtogenerateproducts ofparticular (TdT) (100ngpolyARNA,calfthymus). Gene specificprimers Arabidopsis leaf),andTerminal DeoxynucleotidylTransferase (100 ngtotalRNA,humanliver),Ubiquitin (1µgtotalRNA, Ta targetsbyone-stepRT-PCR. Amplification ofdiverseRNA rget (source): 0.5 kbÐ 3. 2. 1. M. Marker 1 kbÐ 2 kbÐ β β β -actin (1.5kb) -actin (1.0kb) -actin (0.5kb) (3) and Taq DNAPolymerase β -actin (100ngtotalRNA,humanliver),Numb M 1 2 3 4 5 6 7 Figure 1 7. TdT(1.5kb) 6. Ubiquitin(1.5kb) 5. Numb(1.0kb) 4. Numb(0.5kb) (4) , premixed togetheratconcentrations (2) . Thisapproach offers 0ratos$186.00 (50 50 reactions 78350 One-Step RT-PCR Kit µ l/rctn) Amplify 11 M. Marker 1. 0.4µM 2. 0.2µM 3. 0.1µM 4. 0.075µM -actin Notch3 (0.34 kb) β (1.5 kb) Primers were PolyA RNA M. Marker 1. 100 ng 2. 10 ng 3. 1 ng 4. 100 pg 5. 10 pg 6. 1 pg 7. 100 fg ← ← (a) For many targets, such as 1.5 kb Figure 3 -actin and Notch3 from human liver total l RNA β b a Primer + - + - + Tota M. Marker 1. 1 µg 2. 100 ng 3. 10 ng 4. 1 ng 5. 100 pg 6. 10 pg 7. 1 pg concentration - 1 2 3 4 3 2 1 M M 42 42 50 50 -actin (¡C): β (0.5 kb) betaine: ← 2 kb Ð 1 kb Ð 0.5 kb Ð 0.2 kb Ð 0.5 kb Ð 1. 5 M RT temp -actin, specificity may be improved by decreasing the primer concentration. by decreasing -actin, specificity may be improved eaction with supplement and elevated temperature. Highly specific detection of RNA (100 ng), by one-step RT-PCR. β with high (b) For targets for 0.4µM versus 0.075µM primer. results Compare G+C contents, such as 0.34 kb Notch3 (G+C: 77%), adding supplements transcription step, may of the reverse the temperature and/or increasing conditions versus reaction for standard results Compare specificity. improve r Figure 2 Figure RNA PolyA 2 3 4 5 6 7 2 3 4 5 M 1 J. Biol. Chem. www.usbweb.com ) 2 l -actin target from human liver total RNA-actin target from human and polyA one-step RT-PCR. RNA, by β RNA Tota 2 3 4 5 6 7 2 3 4 5 M 1 , 1487-1490. 20 1.0 kb Ð 0.5 kb Ð 0.2 kb Ð , 487-491. used at 0.8µM, a relatively high concentration, in order to achieve high sensitivity. in order high concentration, used at 0.8µM, a relatively Highly sensitive detection of Highly sensitive detection Need more info? Reach [email protected]. 239 , 9326-9335. 260 Mullis, K. B., and Erlich, H. A. (1988) R., Horn, G. T., Science A Laboratory Harbor Laboratory Manual,” Cold Spring 8.46-8.53. Press, Nucleic Acids Res. -PCR Kit, rapid and reliable results are just one step away. are results -PCR Kit, rapid and reliable -PCR Enzyme Mix 5X (includes MgCl -PCR Reaction Buffer, 4. S., Scharf, Saiki R. K., Gelfand, D. H., Stoffel, S. J., Higuchi, 3. (1985) S. P. N., and Goff, Roth, M. J., Tanese, 2. L. N., Coelen, R. J., and MacKenzie, J. S. (1992) Sellner, Ultrapure PCR Nucleotide Mix: 10mM each dATP, dCTP, PCR Nucleotide Mix: 10mM each dATP, Ultrapure dTTP dGTP, Recombinant (4 units/µl) Inhibitor, Magnesium Chloride, 25mM Water (DEPC treated) RNase-Free References: 1. (2001) “Molecular Cloning: J. and Russell, D. W. Sambrook, Convenient One-Tube Format Convenient One-Tube is quick and simple, given that the RT Setting up reactions set up simultaneously and then carried and PCR steps are out sequentially in one tube. This format eliminates the need time and saving and PCR independently, to set up RT eliminating a potential point of contamination. Rapid, Reliable Results Kit is ideal for qualitative analysis of The One-Step RT-PCR of one or a few genes in multiple RNA samples, expression the streamlining analysis of specific RNA splice variants and The kit is and PCR steps simultaneously. optimization of RT also particularly good as a starting point for analysis of new be amplified RNA targets, given that many targets can Additional USB successfully without need for optimization. Script Kit and the RT RT-PCR such as the Two-Step products, PCR Kit are PCR Master Mix or Taq Kit coupled with the Taq applications. With the One-Step useful for other RT-PCR RT Kit Components: RT RT 12 Amplify RT-PCR Kit T wo-Step NEW Phone: 1-800-321-9322 | Fax: 1-800-535-0898 carrying outtheRT (orcDNAsynthesis)stepinonetubeandthePCRanother carrying RT based onM-MLV ReverseTranscriptase can beusedwithRNAsamplesandcustomprimersprepared bystandard approaches. Thekitis The Two-Step RT-PCR Kitcomescompleteandready tousefordiverseRT-PCR applications.Thekit All inOneRT-PCR Kit been designedforuseinallofthesetypesRT-PCR. useful fordetectionofoneorafewtargetsinmultipleRNAsamples.TheUSBTwo-Step RT-PCR Kithas amplifying G+Crichtargets,enablespecificamplificationforothers(Fig.3). protocol adjustments, suchasoptimizationoftheamountprimersoradditionsupplementsfor Standard reaction conditionsare sufficient forspecificamplificationofmosttargets.Afewsimple polyA RNA.Highlyexpressed targetscanbedetectedinevenloweramountsofRNA(Fig.2). weakly expressed targetscangenerallybedetectedin1ngtoµgtotalRNAor100pg (~0.2to1.5kb)PCRproducts (Fig.1).Moderatelyand least 5.6kb)followedbyamplificationofshort The kitcanbeusedfordetectionofdiverseRNAtargetsbasedongenerationlongcDNAs(toat Highly Sensitive,Specific dilution stepalsoallowsuseofthekitforone-stepRT-PCR. free waterare alsoincluded,allowingflexibilityinsettingupRT andPCRindividually. Asimpleenzyme PCR reaction buffers, RNaseinhibitor, Ultrapure dNTPs,supplementalmagnesiumchlorideandRNase- concentrations optimizedtobalancesensitivityandspecificityintwo-stepRT-PCR. OptimizedRT and •A •O •H •Q tube formats. outRT-PCRDesigned forflexibilityandversatilityincarrying inoneortwo- T A third one-stepRT-PCR, approach, oftentermed using oneRT reaction, primedwitholigodT, fordetectionofmultipletargetsfrom thesameRNAsample. specific primers,fordetectionofasingletargetfrom asingleRNAsample.Anotherapproach involves with gene specific primers and carrying outthereactions sequentiallyinonetube with genespecificprimersandcarrying wo-Step RT-PCR Kit -PCR maybecarriedoutinseveralways.Oneapproach, two-stepRT-PCR, oftentermed involves oligo dTprimers nalysis oftheexpression ofmultiplegenesinindividualRNAsampleswith ighly detailedanalysisofRNAsplicevariants ptimization ofPCRindependentRT ualitative analysisofgeneexpression generate cDNAofatleast5.6kb. of 5.6kbClathrintarget(humanliver)byusealong-PCRmethodimplies that RT reaction can used). Genespecificprimerswere sizes.Amplification designedtogenerateproducts ofparticular mlfcto fdvreRAtargetsbytwo-stepRT-PCR. Amplification ofdiverseRNA conducted on10 (TdT) (calfthymus).RT wascarriedouton1µgtotalRNAwithprimingbyoligodT, andPCRwas liver), Numb(humanUbiquitin(Arabidopsisleaf),andTerminal DeoxynucleotidylTransferase 0.5 kbÐ 1 kbÐ 2 kbÐ M -1 dilution ofRT reaction (exceptforTdT, forwhichnon-dilutedRT reaction was 1 2 3 4 5 6 7 M 8 (3) and Taq DNAPolymerase Figure 1

involves settingupthe Ta rget (source): 8. Clathrin(5.6kb) M. Marker 7. TdT(1.5kb) 6. Ubiquitin(1.5kb) 5. Numb 4. Numb 3. 2. 1. M. Marker (4) , provided inindividualtubesat β β β -actin (1.5kb) -actin (1.0kb) -actin (0.5kb) RT β

(1.0 kb) (0.5 kb) and PCR simultaneously -actin (human (2) . This isparticularly (50 (25 100 PCRreactions 50 RTreactionsand 78355 T wo-Step RT-PCR Kit µ µ l/PCR rctn) l/RT rctn) (1) with gene $208.00 Amplify 13 Science M. Marker 1. 0.4µM 2. 0.2µM 3. 0.1µM 4. 0.075µM -actin M. Marker 1. No supplement 2. 1.0M betaine β (1.5 kb) ← Notch3 (0.34 kb) ← (a) For many targets, such as 1.5 kb Figure 3 Figure a Primer -actin and Notch3 from human liver total RNA concentration b 1 2 1 1 2 3 4 3 2 1 β M M , 1487-1490. 20 2 kb Ð 1 kb Ð 0.2 kb Ð 0.5 kb Ð 0.5 kb Ð -actin, specificity may be improved by decreasing the primer decreasing by -actin, specificity may be improved (100 ng), by two-step RT-PCR. Specific detection of β concentration in the PCR step. Compare results for 0.4µM versus for 0.4µM results concentration in the PCR step. Compare (b) For targets with high G+C contents, such as 0.34 kb 0.075µM primer. of the reverse the temperature Notch3 (G+C: 77%), increasing PCR and/or RT transcription step and/or adding supplements for the was carried out at 50°C reaction RT specificity. steps, may improve supplements or without supplements, and PCR was conducted with no generation of in of betaine results with 1.0M betaine. The presence product. desired , 9326-9335. 260 Nucleic Acids Res. -actin β (0.5 kb) ← J. Biol. Chem. www.usbweb.com ) RNA 2 PolyA ) 2 3 4 5 6 2 3 4 5 2 PolyA RNA M. Marker 1. 100 ng 2. 10 ng 3. 1 ng 4. 100 pg 5. 10 pg 6. 1 pg M 1 was carried out in 25 µl volume on indicated

Figure 2 Figure RT -actin target from human liver total RNA and l l RNA β RNA Tota Tota M. Marker 1. 1 µg 2. 100 ng 3. 10 ng 4. 1 ng 5. 100 pg 6. 10 pg 2 3 4 5 6 2 3 4 5 M 1 , 487-491. 1.0 kb Ð 0.5 kb Ð 0.2 kb Ð latively high concentration, in order to achieve high sensitivity. to achieve high latively high concentration, in order Reaction Buffer, 5X (includes MgCl Reaction Buffer, 239 8.46-8.53. Highly sensitive detection of amounts of RNA by use of a gene specific primer. PCR was conducted in 50 µl amounts of RNA by use of a gene specific primer. used at 0.8µM, a For PCR, primers were reaction. volume on 2 µl aliquot of RT re polyA RNA, by two-step RT-PCR. aq DNA Polymerase 4. H. A. (1988) Mullis, K. B., and Erlich, S., Scharf, Saiki, R. K., Gelfand, D. H., Stoffel, S. J., Higuchi, R., Horn, G. T., 3. (1985) S. P. N., and Goff, Roth, M. J., Tanese, 2. L. N., Coelen, R. J., and Mackenzie, J. S. (1992) Sellner, PCR Nucleotide Mix, Ultrapure: 10mM each dATP, dCTP, dGTP, dTTP dGTP, dCTP, 10mM each dATP, PCR Nucleotide Mix, Ultrapure: (4 units/µl) Recombinant Ribonuclease Inhibitor, Magnesium Chloride (25mM) Water (DEPC-treated) RNase-Free References: 1. (2001) “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, J. and Russell, D. W. Sambrook, PCR Reaction Buffer, 10X (includes MgCl PCR Reaction Buffer, RT Flexible Format due to the accessible experiments are A wide range of RT-PCR can be applied of this kit. One set of reagents all-in-one nature in flexibility allowing great easily in multiple types of RT-PCR, planning, carrying out and optimizing experiments. Applications One Kit, Many for diverse RT-PCR Kit is well suited RT-PCR The Two-Step primer formatapplications. The two-step/gene specific is highly expression, excellent for qualitative analysis of gene format The two-step/oligo dT primer is and optimization of PCR independent of RT. detailed analysis of RNA splice variants, And the one-step format of multiple genes in individual RNA samples. excellent for analysis of the expression for is excellent RNA samples. Depending on the scale of experiments and the size of PCR products, rapid analysis of single genes in multiple efficient. PCR Kit may be more PCR Master Mix or Taq Kit coupled with the Taq Script Kit or the RT use of the One-Step RT-PCR many applications. Kit is one kit with RT-PCR The Two-Step Kit Components: Reverse Transcriptase M-MLV T 14 Amplify Kit RT Script NEW Phone: 1-800-321-9322 | Fax: 1-800-535-0898 priming inparticular, theexpression ofhundreds ofgenesmaybeanalyzedfrom asingleRT reaction. based onM-MLV ReverseTranscriptase diverse RNAsamplesandcustomprimersprepared byavarietyofstandard approaches. Thekitis The RT ScriptKitcomescompleteandready tousefortheRT stepofRT-PCR. Thekitcanbeusedwith SynthesisOptimizedforRT-PCR cDNA appropriate RT conditions,diversetargetsandavarietyofapplicationsbecomeaccessible. Successful RT-PCR dependsonuseofappropriate conditionsfortheRT step.Withinthecontextof •U •O Kit orotherUSBPCRreagents canbeusedforthePCRstep. accomplishes theRT stepinRT-PCR. USBTaq PCRMasterMix,theTaq PCR Designed forreverse transcriptionofRNAtoyieldtemplateforPCR.Thekit RT ScriptKit from 10 reagents.PCR Yields from the RT detected readily byuseofTaq PCRMasterMix,typicallywithoutneedforoptimization,orotherUSB be reverse transcribedfrom evenloweramountsofRNA(Fig.2).Theresulting cDNAproducts canbe transcribed from 1ngtoµgtotalRNAor100pgpolyARNA.Highlyexpressed targetscan appropriate forthedesired product (Fig.1).Moderatelyandweaklyexpressed targetscanbereverse use ofeithergenespecificprimersoroligodT. TheRT stepisfollowedbyPCRwithareagent The kitcanbeusedforgeneratinglongcDNA(toatleast5.6kb)from diverseRNAtargets,basedon High Sensitivity, High Yield RT supplemental magnesiumchloride,andRNase-free waterare alsoincluded,allowingsimplesetupof standard RT-PCR applications.AnoptimizedRT reaction buffer, RNaseinhibitor, Ultrapure dNTPs, variants inindividualRNAsamples reactions tobefollowedbyPCR. seful fortheanalysisofexpression ofmultiplegenesorRNAsplice ptimized forgenerationofcDNAspecificallyuseinPCR -1 to 10 -4 dilutions oftheRT reactions, dependingonRNAtargetabundance(Fig.3).WitholigodT Script KitandUSBTaq PCRMasterMix. targetsbytwo-stepRT-PCR withtheRT Amplification ofdiverseRNA r clathrin target(humanliver)byuseofalong-PCR methodimpliesthatRT sizes.Amplificationof5.6kb designed togenerateproducts ofparticular which non-dilutedRT reaction wasused).Genespecificprimers were PCR wasconductedon10 liver). RT wascarriedouton1µgtotalRNAbyprimingwitholigodT, and Deoxynucleotidyl (TdT)(calfthymus),and Clathrin(human (human liver),NumbUbiquitin (Arabidopsisleaf),Terminal eaction cangeneratecDNAofatleast5.6 kb. 0.5 kbÐ 1 kbÐ 2 kbÐ 5. Numb(1.0kb) 4. Numb(0.5kb) 3. 2. 1. M. Marker stepare typicallysufficient toallowPCRdetectionofcDNAproducts β β β M -actin (1.5kb) -actin (1.0kb) -actin (0.5kb) (1) 1 3 5 7 9 M 1 2 3 4 5 6 7 8 provided ataconcentrationoptimizedforsensitivityin -1 dilution ofRT reaction (exceptforTdT, for Figure 1 9. Clathrin(5.6kb) M. Marker 8. Clathrin(3.8kb) 7. TdT(1.5kb) 6. Ubiquitin(1.5kb) Ta rget (source): β -actin 0ratos$145.00 (25 50 reactions 78360 RT ScriptKit µ l/rctn) Amplify 15 sion -actin (1.0 kb) β Numb (1.0 kb) Clathrin (3.8 kb) ← ← ← -4 10 ) of the RT product was product ) of the RT -4 Figure 3 Figure was carried out in a 25 µl volume -3

10 RT to 10 -2 -1 10 -actin. (b) 1.0 kb Numb. (c) 3.8 kb Clathrin. β -1 reaction 10 Dilution of RT 0 10 c a b , 1487-1490. aq PCR Master Mix. generated. PCR was conducted in a 25 µl volume on 1 µl generated. PCR was conducted in a 25 µl volume on and each dilution. Gene specific reaction the RT aliquots from of particular designed to generate products primers were sizes. (a) 1.0 kb Detection of multiple RNA a dilution series of targets from of the RT Script Kit and USB a single RT reaction, by use T on 1 µg total RNA from human liver with priming by oligo dT. on 1 µg total RNA from A dilution series (10 20 , 9326-9335. 260 Nucleic Acids Res. -actin β (0.5 kb) ← J. Biol. Chem. www.usbweb.com RNA PolyA 2 3 4 5 6 2 3 4 5 ) 2 PolyA RNA M. Marker 1. 100 ng 2. 10 ng 3. 1 ng 4. 100 pg 5. 10 pg 6. 1 pg Order online at www.usbweb.com. M 1 Figure 2 Figure l l RNA -actin target from human liver total RNA and polyA β RNA Tota Tota M. Marker 1. 1 µg 2. 100 ng 3. 10 ng 4. 1 ng 5. 100 pg 6. 10 pg 2 3 4 5 6 2 3 4 5 M 1 1.0 kb Ð 0.5 kb Ð 0.2 kb Ð was carried out in a 25 µl volume on indicated amounts of RNA by use of a gene was carried out in a 25 µl volume on indicated amounts

Reaction Buffer, 5X (includes MgCl Reaction Buffer, 8.46-8.53. Highly sensitive detection of RNA, by two-step RT-PCR with the RT Script Kit and USB Taq PCR Master Mix. with the RT Script Kit and USB Taq RNA, by two-step RT-PCR RT specific primer. PCR was conducted in a 25 µl volume on 1 µl aliquot of RT reaction. For reaction. PCR was conducted in a 25 µl volume on 1 µl aliquot of RT specific primer. to achieve in order high concentration, used at 0.8µM, a relatively PCR, primers were high sensitivity. equired, or, in the case of the Taq PCR Master Mix, where simplicity and convenience in set up of PCR are highly valued. simplicity and convenience in set up of PCR are PCR Master Mix, where in the case of the Taq or, equired, 3. L. N., Coelen, R. J., and MacKenzie, J. S. (1992) Sellner, 2. (2001) “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, J. and Russell, D. W. Sambrook, PCR Nucleotide Mix, Ultrapure: 10mM each dATP, dCTP, dGTP, dTTP dGTP, dCTP, 10mM each dATP, PCR Nucleotide Mix, Ultrapure: (4 units/µl) Recombinant Ribonuclease Inhibitor, Magnesium Chloride (25mM) Water (DEPC treated) RNase-Free References: 1. (1985) S. P. N., and Goff, Roth, M. J., Tanese, RT-PCR with a Simple Start and Flexible Finish with a Simple Start and Flexible RT-PCR of genes, depending on the choice of Script Kit can be used to generate cDNA for individual genes or a distribution The RT most appropriate type and amount of reagent step is followed by PCR using the RT The all based on a single protocol. primer, start with a simple and flexible finish. the benefit of RT-PCR Script Kit offers for a given application. Thus, the RT RT for PCR is useful for the analysis of expres Script Kit is optimized for generation of cDNA specifically for use in PCR. The kit The RT of multiple genes or RNA splice variants in individual RNA samples. This is particularlyof multiple genes or RNA splice variants are many targets true in cases where substantial optimization of PCR is where reagents, PCR different best amplified by targets are different analyzed, where r Kit may also be RT-PCR Two-Step Kit or aims, the One-Step RT-PCR Depending on specific targets and experimental for PCR. RT Script Kit achieves excellent The RT appropriate. Kit Components: Reverse Transcriptase M-MLV RT 16 Amplify SSB Phone: 1-800-321-9322 | Fax: 1-800-535-0898 DNA forsubsequentmutagenesis target restriction endonucleasedigestionstospecificsitesinsingle-stranded libraries ofdouble-strandedDNA carrying outsite-directed mutagenesis carrying 4. Sigal, N., Delius, H., Kornberg, T., Sigal,N.,Delius,H.,Kornberg, Gefter, B.(1972) M.L.andAlberts, 4. S.andGriffith, Chrysogelos, J.(1982) 3. H.,Schomburg,U.andMaass,G.(1981) KraussG.,Sindermann, 1. References: glycerol. 20mM Tris-HCl (pH7.5),200mMNaCl,0.1mMEDTA, 1.0mMDTT, 50% Storage Buffer: 1- 5µg/µl Concentration: Polymerases usedinDNAsequencingreactions pyrosequencing forSNPanalysis structure. More recently, SSBwasusedtohelpobtainlongerread lengthsin and eliminatespausingwhensequencingthrough strong secondary microscopy protein hasbeenusedtovisualizesingle-strandedDNAbyelectron DNA manner tosingle-strandedDNAanddoesnotbindwelldouble-stranded Single-Stranded DNABindingProtein bindswithhighaffinity inacooperative BindingProtein(SSB) Single-Stranded DNA SSB hasbeenshowntobeeffective influorescence polarizationassays .Honigberg,S.M.,Rao,B.J.andRadding,C.M. (1986) 7. D.(1980) Shortle, 6. Muskavitch,K.M.andLinn,S.(1982) 5. Weiner, A.(1975) L.andKornberg, J.H.,Bertsch, 2. 3 Chase,J.W. andWilliams,K.R.(1986) 13. Hsu,T. M.,Chen,X.,Duan,S.,Miller, R.D.andKwok,P.10. Y. (2001) Milavetz,B.(1989) 9. Kowalczykowski,S.C.,Bear, D.G.andVon Hippel,P. H.(1981)inThe 8. 4 Williams,K.R.,Spicer, E.K.,Lopresti, M. B.,Guggenheimer, R.A.,and 14. 12. Ronaghi,M.(2000) 11. Andreasson, A., Asp, A., Alderborn, A.,Gyllensten, U.andAllen,M. Andreasson, A.,Asp,Alderborn, 11. 5 Griffith, J.D.,Harris,L.D.andRegister, J.(1984) 15. 7 Sancar, A.,Williams,K.R.,Chase,J.W. andRupp,W.17. D.(1981) Julin,D.A.,Riddles,P. W. andLehman,I.R.(1986) 16. Proc. Natl.Acad. Sci. 5803-5807. 1972-1980. Biochemistry Sci. BioTechniques 373. Enzymes, 3rd edition, ed.P. D.Boyer, (AcademicPress, NewYork) 14, Chase, J.W. (1983) (2002) Symposia onQuant.Biol 1025-1030. Natl. Acad.Sci. (1,2) . ItisinvolvedinDNAreplication andinrecombination USA BioTechniques (3,4,5) 83 . IthasalsobeenusedinconjunctionwithRecAprotein for , 9586-9590. 20 31 , 5346-5352. USA , 560-570. Proc. Natl.Acad. Sci. Nucl. AcidsRes. Anal. Biochem J. Biol.Chem. 78 USA 2(1) 32 , 4274-4278 . 49 69 , 124-133. , 553-559 (7) , 3537-3541. (11,12) (9) . SSBmayalsostimulatespecificDNA . . 258 (6) 286 17 and toselectsequencesfrom J. Biol.Chem. Proc. Natl.Acad. Sci. , 3346-3355. , 282-288. Ann. Rev. Biochem. , 3322. USA (8) 77 and hasbeenusedto , 5375-5379. Cold SpringHarbor J. Biol.Chem. 257 J. Biol.Chem. Proc. Natl. Acad. , 2641-2648. in vivo 55 USA , 103-136. Proc. . This 79 261 250 , (10) , , 500 70032Z 100 70032Y Binding Protein(SSB) Single-Stranded DNA µ µ g g$ $337.00 84.00 Amplify 17 84.00 $337.00 $227.00 $603.00 l) l) µ µ g g/ g/ µ µ µ g$ g$ g g µ µ µ 10 ≥ T4 Gene 32 Protein T4 Gene 32 Concentration Standard (1 - 5 70029Y 100 70029Z 500 High Concentration ( 74029Y 300 74029Z 1,000 62, , 7251- , 89-104. , 341-350. , 103-136. , 415-417. Nucl. Acids 88 251 67 55 , 693-702. 260 J. Mol. Biol. 96 Nucleic. Acids , 12274-12279. . , 267-271. (7) 258 62 J. Mol. Biol. J. Mol. Biol. J. Biol. Chem. is required in stoichiometric is required J. Mol. Biol. . On a primed single-stranded J. Biol. Chem. (5,6) . It has been noted to eliminate . It has been noted to in vivo Ann. Rev. Biochem. Ann. Rev. J. Mol. Biol. (3,4) , 8844-8848. J. Biol. Chem. 83 . It also binds to single-stranded RNA (with to single-stranded . It also binds USA (1,2) www.usbweb.com , 6426-6432. 254 . , E29. (8) (5) , 1079. Proc. Natl. Acad. Sci. Proc. lower affinity than single-stranded DNA) allowing it to control its own allowing it to control single-stranded DNA) than lower affinity 18 29 4 - 10 (1986) J. Biol. Chem. Res. 39-52. Res. 7262. 1 equired for T4 DNA replication, recombination and repair. It binds and repair. recombination DNA replication, for T4 equired 13. H., Williams, K. R. and Chase, J. W. W. Adari, H., Konigsberg, Y., Shamoo, 12. M. D. and Sinha, N. K. (1983) Topal, 11. and Morgan, A. R. (1985) D. F. Dombroski, 9. H. (1979) E. and Konigsberg, W. D. K. R., Sillerud, L. O., Schafer, Williams, rate of synthesis at the level of translation rate of synthesis at the 10. and Bartram, C. (1990) K., Hansen-Hagge, T. Schwarz, T4 Gene 32 Protein has been shown to be effective in stimulating PCR. The in stimulating been shown to be effective has T4 Gene 32 Protein in RNA to enhance yield and processivity enzyme has been found amplification pausing when sequencing through strong secondary strong structure. through pausing when sequencing been widely used in studies of DNA-protein has T4 Gene 32 Protein of single-stranded DNA in cytological regions interactions and for marking microscopy by electron viewed preparations in a 5 - 10-fold results of T4 Gene 32 Protein DNA template the addition by T4 DNA Polymerase in the rate of synthesis increase T4 Gene 32 Protein T4 Gene 32 which is protein DNA binding is a single-stranded Protein T4 Gene 32 r DNA and to single-stranded cooperatively rather than catalytic quantities rather than 7. Huberman, J. A., Kornberg, A. and Alberts, B. M. (1971) 6. R. (1975) A. and Yuan, Bickle, T. Brack, C., 8. (2001) C. P. E. L. and Hunter, L. R., Hill, A. A., Brown, Baugh, 5. H., Mantell, N. J., and Alberts, Delius, B. (1972) 4. Z., and Russel, M. (1976) P. L., O’Farrel, Gold, 3.H. M., Bolle, A. and Epstein, R. H. (1974) Krisch, 2. (1971) K. and Snustad, D.P. Sinha, N. 10 Storage Buffer: 50% 0.5mM DTT, (pH 8.0), 100mM NaCI, 1.0mM EDTA, 20mM Tris-HCI glycerol. References: 1. and Williams, K.R. (1986) J. W. Chase, T4 Gene 32 T4 Gene Protein Order online at www.usbweb.com. Ask about our customerization program. Don’t see the concentration or the size you need? Don’t see the concentration or the size you need? 18 Amplify Heat-Labile UNG, UNG Phone: 1-800-321-9322 | Fax: 1-800-535-0898 .Longo,M.C.,Berninger, M.S.andHartley, J.L.(1990) 2. previous reactions r Uracil-DNA Glycosylasecanbeutilizedfortheprevention offalsepositive of PCRproducts havingdUcontainingprimersincorporatedintothem Uracil-DNA Glycosylasecanalsobeusedtoincrease thecloningefficiency dU-rich PCRproducts. inactivated duringsubsequenttemperature cyclingandwillnotaffect thenew dU isunaffected andwillbeamplifiednormally. Uracil-DNAGlycosylaseis contaminants preventing amplification.Thetemplatewhichdoesnotcontain Glycosylase results inexcisionofuracilfrom thedU-containingcarry-over reactionsPre-incubation withUracil-DNA ofsubsequentPCRstarting dTTP inthePCRreaction mixtoincorporatedUTPinallofthePCRproducts. well asincreasing theefficiency ofsite-directed mutagenesis DNA Glycosylase whereas RNA and normal dT-containingDNA Glycosylasewhereas RNAandnormal DNAare not Double andsingle-strandeddU-containingDNAare substratesforUracil- hybridization site. blocked from replication byDNApolymeraseorprevented from becominga backbone. Thiscleavagegeneratesalkalisensitiveapyrimidinicsitesthatare cleaving theN-glycosidicbondbetweenuracilbaseandsugar Uracil-DNA Glycosylaseexcisesdeoxyuracilfrom dU-containingDNAby Glycosylase Uracil-DNA .Lindahl,T., W., Ljungquist,S.,Siegert, Nyberg,B.andSperens, B.(1977) 1. References: 50mM HEPES-KOH(pH7.4),1mMDTT, 1mMEDTA,10mM NaCl. GlycosylaseDilutionBuffer(1mlincluded,PN71961): Uracil-DNA 30mM Tris-HCl (pH7.5),150mMNaCl,1mMEDTA, 1mMDTT, 50%glycerol. Storage Buffer: 1 unit/µl Concentration: .Kunkel,T. (1985) 4. Nisson,P. E.,Rashtchian,A.andWatkins, P.3. C.(1991) 50% glycerol. 12mM Tris-HCl (pH8.0),70mMNaCl,0.5mMEDTA, 50µg/mlBSA, Storage Buffer: 1 unit/µl Concentration: incubation at50°C. heat-labile. Itiscompletelyandirreversibly inactivatedafter10min attributes oftheenzymederivedfrom This Uracil-DNAGlycosylase**,derivedfrom Glycosylase,Heat-Labile Uracil-DNA esults in PCR caused by trace amounts of carry-over contaminantsfrom esults inPCRcausedbytraceamountsofcarry-over J. Biol.Chem. 120-123. 252 (2) Proc. Natl.Acad.Sci.USA . ThiscanbeaccomplishedbysubstitutingdUTPfor , 3286-3294. E. coli , withtheaddedbenefitofbeing 82 Gadus morhau , 488-492. PCR MethodsAppl. Gene , hasallthe (4) 93 . , 125-128. (3) (1) as . 1 , 0 nt $78.00 100 units 78310 Heat-Labile Glycosylase, Uracil-DNA l$18.00 1 ml 71961 Dilution Buffer $67.00 Glycosylase Uracil-DNA 100 units 71960Y Glycosylase Uracil-DNA Amplify 19 Price . 2 l. µ ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE 2 20 units/ ≥ www.usbweb.com 25 ml1 lt $ 16.00 $ 58.00 5 x 50 mg5 x 1 ml $180.00 $ 34.00 B Grade torage Buffer: 20mM HEPES-KOH (pH 7.6), 50mM KCl, 8mM DTT, 20mM HEPES-KOH (pH 7.6), 50mM KCl, 8mM DTT, torage Buffer: unctionally tested in PCR unctionally tested in PCR unctionally tested in PCR unctionally tested in PCR unctionally tested in PCR ested User Friendly ested User Friendly ested User Friendly ater, PCR Qualified ater, MB Grade The 10X Buffer consists of 100mM Tris (pH 8.6), 500mM KCl. Tris consists of 100mM The 10X Buffer The 10X Buffer consists of 100mM Tris-HCl (pH 8.6), 500mM KCl, 15mM MgCl Tris-HCl consists of 100mM The 10X Buffer Free of contaminating Free of contaminating 50% glycerol with a concentration of W ¥ ¥F 71785 10 x 1 ml $32.00 7116510X PCR Reaction Buffer without MgCl 1 ml¥ ¥T ¥F 71166 Recombinant RNase Inhibitor, 1 ml¥S 71571 5,000 units $19.00 $19.00 $125.00 Mineral Oil, Light ¥M 71600 10 ml 10X PCR Reaction Buffer ¥ ¥T ¥F $ 11.00 10921 50 mgGelatin ¥F 70086 25mM Solution Magnesium Chloride, 100 gm¥T ¥F 71167 1 ml $42.00 $ 22.00 $ 16.00 Prod. No.Solution Bovine, 50 mg/ml Albumin, Size ¥ Don’t see the concentration or the size you need? Don’t see the concentration or the size you need? Ask about our customerization program. PCR Related Reagents 20 Purify ExoSAP-IT ¨ Phone: 1-800-321-9322 | Fax: 1-800-535-0898 ExoSAP-IT and within30minutesbeready tosequenceordoSNPanalysis. ExoSAP-IT consistsofonepipettingstepandtwoincubations.JustaddtothePCRproduct Rapid PCRProductClean-UpProtocol PCR treatment, ExoSAP-ITisinactivatedsimplybyheatingto80°Cfor15minutes. ExoSAP-IT isaddeddirectly tothePCRproduct andincubatedat37°Cfor15minutes(Fig.1).After PCR. ShrimpAlkalinePhosphataseremoves theremaining dNTPsfrom thePCRmixture. re buffer,a speciallyformulated toremove unwanteddNTPsandprimersfrom PCRproducts. ExonucleaseI ExoSAP-IT utilizestwohydrolytic enzymes,ExonucleaseIandShrimpAlkalinePhosphatase,togetherin • • • • • • Polymorphism (SNP)analysis. downstream applications,suchasDNAsequencingorSingleNucleotide ExoSAP-IT reagent isdesignedforsimple,quickPCRclean-up moves residual single-strandedprimersandanyextraneousDNAproduced inthe and plates Simple Processing Scaleable downstream applications Removes ContaminatingPrimersanddNTPs products Conserves PCRSamples increasing yield Eliminates SpinColumns product One Tube/One StepPCRClean-Up ¨ For PCRProductClean-Up — Economicalforhighthroughput purification — Lendsitselftorobotics; Replacesbeads,filtrations Summary ofExoSAP-ITPCRproduct treatment. — 100% recovery of both short andlongPCR ofbothshort — 100%recovery — Decreases timeandexpensewhile — AddExoSAP-ITdirectly toPCR Figure 1 — No interference in — Nointerference Request Custom FormulationsBy $2,600.00 5,000 reactions 78205 $1,105.00 2,000 reactions $321.00 78202 500 reactions $80.00 78201 100 reactions 78200 Product Clean-Up ExoSAP-IT ForPCR Purify 21 , 537- 30 PCR clean-up , 811- 818. BioTechniques Figure 2 Figure 47 , 323-326. 418 , 4354-4355. 22 Nature J. Forensic Sci J. Forensic 2a 2b Fluorescent sequencing results of a 100 bp pUC18 PCR Fluorescent sequencing results of a 100 bp pUC18 fragment sequenced with a Ð20 Fwd primer using fluorescent sequencing reagents. (b) a column designed for performed with: (a) ExoSAP-IT; low due to inherently PCR clean-up. Base miscalls in (b) are yields of short when using columns. PCR products Nucleic Acids Res. . There is . There (1) , 858-860. 17 www.usbweb.com BioTechniques may be formulated to meet your specific needs. ¨ 542. Monteiro, E., De Jesus, A., Holanda, M. A., Zago, M. A., Simpson, A. J. G. and Neto, E. D. (2001) De Jesus, A., Holanda, M. A., Zago, M. A., Simpson, A. J. G. and Neto, E. D. (2001) E., Monteiro, 5. Werle, E., Scneider C., Renner, M., Volker, M. and Fiehn, W. (1994) 5. Fiehn, W. M. and M., Volker, E., Scneider C., Renner, Werle, ExoSAP-IT Simple: Single-Step of ‘hands-on’ time. a minimum is designed to require The method nucleotides and unincorporated of excess primers removal Enzymatic a single tube or in ExoSAP-IT reagent easy step by using occurs in one many therefore, required, pipette transfers are well. Only simple microtiter at once, either manually or with robotic samples can be processed devices. Quality from PCR Products Achieve High Data primers and nucleotides yields excess to remove Enzymatic treatment PCR with left-over be easily analyzed. Problems templates which can virtually are eliminated. ExoSAP-IT background primers leading to high or clean-up method prior to fluorescent may be used as an effective (Fig. 2), SNP analysis or any other radioactive DNA sequencing and of excess nucleotides free a PCR product application requiring primers. No Sample Loss purifications, Use of ExoSAP-IT eliminates all gel or column magnetic separations sedimentations, filtrations, beads and/or 4. Silva, Jr., W. A., Costa, M. C. R., Valente, V., De Freitas Sousa, J., Pacó-Larson, M. L., Espreafico, E. M., Camargo, S. S., Sousa, J., Pacó-Larson, M. L., Espreafico, De Freitas 4. V., A., Costa, M. C. R., Valente, W. Jr., Silva, 100% recovery of both short with ExoSAP-IT. and long PCR products Shipping and Storage Shipped on dry at -20°C. ice. Store References: 1. Budowle B. (2002) C. L. and D. R., Fisher, H. S., Hares, K. A., Lawrence, Dugan, 2. M. and Wink, M. (1994) Hanke, 3. and Su X. (2002) C., Branch, O., Li, W. D., Huynh, J., Duan, J., Makova, K., Joy, Mu, 22 Purify A-Taq-IT NEW ª Phone: 1-800-321-9322 | Fax: 1-800-535-0898 MgCl includes twoPCRbuffers forstandard reactions, onewithandwithout are functionallytestedinstandard PCRtoensure reliable results. Thekitalso including Ultrapure PCR-QualifiedWater andUltrapure PCRNucleotideMix, purification canbeaccomplishedbyusingjustonekit.Allcomponents, By combiningUSBTaq DNAPolymerasewithExoSAP-IT, amplificationand primers andnucleotides. any otherapplicationwhichwouldbeadverselyaffected bytheleftover ExoSAP-IT, thePCRproduct issuitableforDNAsequencing,SNPanalysisor products. Oncethecontaminatingprimersandnucleotidesare removed by ofPCR the needforspincolumnsorfiltrationsandyields100%recovery nucleotides present inthePCRproduct mixture. UseofExoSAP-ITeliminates ExoSAP-IT isanovelproduct thatdegradestheunusedprimersand exonuclease activities. tested forfunctionandhasnodetectablecontaminatingendonucleaseor polymerase activityanda5' products. Taq DNAPolymerasepossessesahighlyprocessive 5' one kittooffer acompletesolutionforamplificationandpurificationofPCR The A-Taq-IT PCRKitcombinesUSBTaq DNAPolymeraseandExoSAP-ITin 10X PCRBuffer withoutMgCl 10X PCRBuffer 10mM PCRNucleotideMix,Ultrapure T Kit Components: conditions. ExoSAP-IT PCR-Qualified Water, Ultrapure 25mM MgCl A-Taq-IT aq DNAPolymerase,250units Order onlineatwww.usbweb.com. 2 , andaseparatetubeof25mMmagnesiumchlorideforoptimizingPCR ª PCR Kit 2 → 2 3' exonucleaseactivity. USBTaq isrigorously → 3' (100 100 PCRreactions 100 ExoSAP-ITreactions 71175 PCRKIT A-Taq-ITª µ l/rctn) $227.00 Analyze 23 -Agarase I -Agarase -Agarase Reaction Shrimp Alkaline (SAP) 70092Y 500 units70092Z $ 64.00 1,000 units70092X $111.00 5,000 units $380.00 SAP 10X Reaction Buffer, 70103 1 mlSAP Dilution Buffer 72761 $18.00 1 ml $18.00 73430Y 100 units73430Z $ 74.00 500 units $306.00 10X Buffer, 73432 1 ml $18.00 β β Nucleic , (No. 1), 17 Comments , 582-585. , 858-860. , 50% glycerol. , 50% glycerol. 2 17 324 Nature . , SNP analysis or labeling methods. 2 , 987-993. P]ATP and T4 Polynucleotide Kinase P]ATP 3 32 §(1) - , 0.1mM ZnCl γ 2 BioTechniques (PN 78200), which includes both enzymes † (PN 78334). Dephosphorylation also prevents -Agarase Reaction Buffer -Agarase Reaction P using [ β ™ 32 www.usbweb.com Can. J. Microbiol. , 4354-4355. 22 United States Biochemical Corporation, Cleveland, OH. Acids Res. pically, the excess dNTPs remaining after PCR interfere with subsequent the excess dNTPs remaining pically, ligation of linearized plasmid DNA in cloning experiments. Shrimp Alkaline ligation of linearized plasmid DNA in cloning -Agarase I -Agarase embedded in DNA fragments is useful in recovering -Agarase subunit to cleaving the agarose by digests agarose -Agarase eaction products, the use of alternativeeaction products, purification methods, such as enzymatic reactions involving DNA synthesis. The hydrolytic properties of involving DNA synthesis. The hydrolytic enzymatic reactions the Shrimp dephosphorylates dNTPs from all remaining in one easy step. When combined with Exonuclease I (PN PCR mixture single-stranded DNA primers and extraneous of residual 70073) for removal r completely eliminated. For are columns, gels or magnetic separations, to ExoSAP-IT convenience, refer Ty low-melting-temperature agarose. agarose. low-melting-temperature neoagaro-oligosaccharides. Concentration: 1 unit/µl Storage Buffer: 50% glycerol. (pH 6.5), 1mM EDTA, 50mM Bis-Tris-HCl 10X Functionally Tested SAP Dilution Buffer (1ml included, PN 72761): (pH 8.0). 50mM Tris-HCl References: 1. (1990) C. W. Ruan, C. C., Samols, S. B. and Fuller, in a ready-to-use format.in a ready-to-use Concentration: 1 unit/µl Storage Buffer: (pH 7.5), 1mM MgCl 25mM Tris-HCl 2. Hanke, M. and Wink, M. (1994) 3. Werle, E., Scneider C., Renner, M., Volker, M. and Fiehn, W. (1994) 3. M. and Fiehn, W. M., Volker, C., Renner, E., Scneider Werle, Shrimp Alkaline Phosphatase (SAP) Shrimp heat-labile alkaline activity, Shrimp Alkaline Phosphatase is a high specific used for Alkaline are phosphatase useful in many applications. the dephosphorylation of 5'-phosphorylated ends of DNA or RNA for subsequent labeling with re the same specific activity as the calf intestine Phosphatase has approximately 9.6) but, unlike the calf enzyme, it is enzyme (800 - 1000 units/mg at 25°C, pH inactivated by heating for 15 min at 65°C. No completely and irreversibly is necessary. further treatment Shrimp Alkaline Phosphatase is particularly PCR products useful in preparing for applications involving sequencing β β β Functionally Tested 10X SAPFunctionally Tested 70103): Reaction Buffer (1ml included, PN (pH 8.0), 100mM MgCl 200mM Tris-HCl (PN 70031) or OptiKinase 2. M. and Lehrach, H. (1986) Burmeister, (1 ml included, PN 73432): (1 ml included, PN (pH 6.5), 10mM EDTA. 100mM Bis-Tris-HCl References: 1. (1957) W. Yaphe, -Agarase I -Agarase SAP β ENZYMES 24 Analyze ENZYMES Ask aboutourcustomerizationprogram. Don’t seetheconcentrationorsize youneed? Shrimp DNase CIAP Phone: 1-800-321-9322 | Fax: 1-800-535-0898 .Baunoch,D.A.,Xu,M., Tewari, A.,Christmas,J.K.andLane,M.A.(1992) 3. P. Abdolrazaghi, Z.andButterworth, (1983) 2. .Kunitz,(1950) 1. References: 200mM Tris-HCl (pH8.5),100mMMgCl ReactionBuffer(1mlincluded,PN70081): Functionally Tested 10XCIAP 25mM Tris-HCl (pH6.9at0°C),2.5mMMgCl Storage Buffer: 2 units/µl Concentration: RT-P Shrimp DNasemaybeusedtoeliminateDNAfrom RNApreparations priorto leaving ssDNAmostlyintact. dsDNA, thustheenzymecanbeusedtospecificallydegrade DNA. Single-strandedDNA(ssDNA)ishydrolyzed atarateof2-5%that heat labileandhasastrong preference forthehydrolysis ofdouble-stranded has approximately 30timesgreater specificactivity. Also,ShrimpDNaseis ShrimpDNaseissimilartoIbut 5'-phosphate and3'-hydroxyl termini. phosphodiester linkagesinDNAtoyielddi-andoligonucleotideswith Shrimp Deoxyribonuclease(DNase)isanendonucleasethatcleaves Shrimp Deoxyribonuclease(DNase) 25mM Tris-HCl (pH7.0),5mMMgCl Storage Buffer: 20 units/µl Concentration: gel purificationorbyspincolumn. extraction,by Alkaline Phosphatasecanbeinactivatedbyphenol:chloroform re (PN 70031)orOptiKinase subsequent labelingwith endsofDNAorRNAfor of5'-phosphorylated used forthedephosphorylation phosphomonoesters, including5'-nucleotides,RNAandDNA.Itiswidely Calf IntestinalAlkalinePhosphatasecatalyzesthehydrolysis ofmany Calf Intestinal Alkaline Phosphatase(CIAP) .Zhanga,Y., Zhua,Y. andZhoub,H.(2000) 1. References: 50mM Tris-HCl (pH8.0). DilutionBuffer(1mlincluded,PN72761): CIAP ligation oflinearizedplasmidDNAincloningexperiments.CalfIntestinal Oncogene (8) CR. , 887-894. , 7 (11) J. Gen.Physiol. , 2351-2353. 32 ™ P using (PN 78334). Dephosphorylation alsoprevents (PN 78334).Dephosphorylation 33 , γ 349. 2 - , 0.1mMZnCl 32 P] ATP andT4PolynucleotideKinase 2 . 2 Int. J.Biochem.Cell.Biol. , 0.25mMCaCl Enzyme 2 , 50%glycerol. 3 , 12-20. 2 , 50%glycerol. 1:32 l$18.00 1 ml $18.00 72761 DilutionBuffer, 10X CIAP 1 ml 70081 ReactionBuffer, 10X $91.00 CIAP 1,500 units 70034Y Phosphatase (CIAP) Calf Intestinal Alkaline 0 nt $72.00 100 units 78305 (DNase) Deoxyribonuclease Shrimp Analyze 25 Exonuclease I Exonuclease 70073Z 2,500 units70073X $ 64.00 5,000 units $119.00 Lambda Exonuclease 27-0865-01 500 unitsLambda Exonuclease $44.00 10X Reaction Buffer, 9865 1 ml $18.00 Nucleic , 8646- 254 5' direction, . Typically, the . Typically, → , 1849-1860. (3) J. Biol. 19. , 858-860. 247 . 17 (1) . 2 J. Biol. Chem. . BioTechniques (2) . In addition, DNA helicase activity can be . In addition, DNA helicase (1) www.usbweb.com , 4354-4355. 22 Acids Res. 8652. Smith, J. A. and Struhl, K., (1987) Current Protocols in Molecular Biology Protocols Smith, J. A. and Struhl, K., (1987) Current (John Wiley and Sons, Inc.). 672-678. leasing 5'-mononucleotides and leaving the terminal and leasing 5'-mononucleotides intact. 5'-dinucleotide 3. M. and Wink, M. (1994) Hanke, (1994) 4. Fiehn, W. M. and M., Volker, E., Scneider C., Renner, Werle, excess primers and any other extraneous single-stranded DNA present in present any other extraneous single-stranded DNA excess primers and involving with subsequent enzymatic reactions will interfere PCR products properties I degrade all single- of Exonuclease hydrolytic DNA synthesis. The used to be allowing the product in the PCR mixture stranded DNA present When combined with Shrimp Alkaline in other applications. efficiently more for dNTP dephosphorylation,Phosphatase (PN 70092) the use of alternative are such as columns, gels or magnetic separations, purification methods, completely eliminated. Concentration: 10 units/µl Storage Buffer: 50% 5mM 2-mercaptoethanol, (pH 7.5), 0.5mM EDTA, 20mM Tris-HCI glycerol. References: 1. and Linn, S. (1972) P.J. Goldmark, 2. K. M. and Linn, S. (1979) J., Telander, Rosamond, Concentration: 5-10 units/µl Storage Buffer: 500 µg/ml BSA, 50% glycerol. (pH 7.6), 1mM DTT, 10mM Tris-HCl 10X Lambda Exonuclease Reaction Buffer (0.5 ml included, PN 9865): 0.67M glycine-KOH (pH 9.3), 25mM MgCl References: 1. D. D., Seidman, J. G., R., Kingston, R. E., Moore, M., Brent, Ausubel, F. 2. A. D. (1967) J. Biol. Chem. 242, Lehman, I. R. and Kaiser, Little, J. W., 3. Higuchi, R. G. and Ochman, H. (1989) Nucl. Acids Res. 17, 5865. Lambda Exonuclease which preferentially Lambda Exonuclease is an exodeoxyribonuclease degrades the 5'-phosphorylated strand of double-stranded DNA. A by using into one strand of a PCR product 5' phosphate can be introduced one phosphorylated primer and one dephosphorylated primer during amplification. The phosphorylated with Lambda strand is then removed DNA for sequencing. Lambda Exonuclease, thus generating single-stranded of duplex hydrolysis processive Exonuclease catalyzes the stepwise and 5'-phosphorylDNA from termini liberating 5' monoucleotides. Lambda terminiExonuclease will not degrade 5'-hydroxyl Exonuclease I Exonuclease 3' DNA in the single-stranded I hydrolyzes Exonuclease re terminus if the 3' proceed and cannot is is processive Hydrolysis phosphorylated. the endonucleolytic to measure I can be used Exonuclease with an DNA reacted single-stranded circular covalently closed cleavage of of interest measured utilizing Exonuclease I utilizing Exonuclease measured Exonuclease I is particularly useful in preparing the products of PCR for Exonuclease I is particularly the products useful in preparing sequencing or labeling methods applications involving Lambda Exonuclease Exonuclease I Exonuclease ENZYMES 26 Analyze ENZYMES Ask aboutourcustomerizationprogram. Don’t seetheconcentrationorsizeyouneed? Exonuclease T7 Gene6 Phone: 1-800-321-9322 | Fax: 1-800-535-0898 .Engler, M.J.andRichardson, C.(1983) 6. Nikiforov, T. T.,5. Rendle,R.B.,Goelet,P., Rogers,Y. H.,Kotewicz,M.L., Ausubel,F. M.,Brent, R.,Kingston, R.E.,Moore, D.D.,Seidman,J.G.,Smith, 3. 4. Shon, M., Germino, J.andBastia,D.(1982) Shon,M.,Germino, 4. .Thomas,K.R.andOlivera,B.M.(1978) 2. 50mM KPO Storage Buffer: 50 units/µl Concentration: processivity anditwillremove termini both5'-hydroxyl and5'-phosphoryl mononucleotides. However, unlikeLambdaExonuclease,theenzymehaslow the stepwisehydrolysis liberating5' ofduplexDNAfrom the5'termini T7 Gene6ExonucleaseissimilartoLambdainthatitcatalyzes from andRNA the5’-termini RNA:DNAhybridsfrom the5’ also degradesnucleotidesatthegapsandnicksofdouble-strandedDNA for sequencing This enzymehasbeenusedforgeneratingsingle-strandedDNAtemplates but isunabletodegradeeitherdouble-strandedorsingle-strandedRNA. 200mM Tris-HCl (pH7.5),100mMMgCl included, PN70992): Functionally Tested 5XT7Gene6ExonucleaseReactionBuffer(1ml It alsodegradesRNAandDNAfrom RNA:DNAhybridsinthe5' liberating mononucleotidesuntilabout50%oftheDNAisacidsoluble 5’ T7 Gene6Exonucleasehydrolyzes duplexDNAnon-processively inthe T7 Gene6Exonuclease .Kerr, C.andSadowski,P. D.(1972) 1. References: → (20) (1994) Anderson, S.,Trainor, G.L.andKnapp,M.R. J. A.ANDStruhl,K.,(1987) Wiley andSons,Inc.). 3’ direction or5’-hydroxyl from nucleotidesby both5’-phosphoryl , 4167-4175. 4 Order onlineatwww.usbweb.com. (pH 6.5),1mMEDTA, 1mMDTT, 50%glycerol. (4) or SNPanalysis Current Protocols inMolecularBiology (5) . J. Biol.Chem. 2 , 250mMNaCl. J. Biol.Chem. J. Biol.Chem. J. Biol.Chem. 247 Nucl. AcidsRes , 253 311-318. 258 257 , 424-429. , → 11197-11205. , → 13823-13827. 3’ direction. 3' direction (John 22, (1,2) (3) . It . l$18.00 1 ml 70992 Reaction Buffer, 5X T7 Gene6Exonuclease $240.00 10,000 units $61.00 70025Z 2,000 units 70025Y T7 Gene6Exonuclease Analyze 27 anscriptase anscriptase AMV Reverse Tr 70041Y 200 units70041Z $ 47.00 1,000 units $193.00 AMV RT Reaction Buffer, 5X 76218 1 ml $18.00 M-MLV Reverse M-MLV Tr 70456Y 25,000 units $121.00 70456Z 100,000 units $385.00 Reaction Buffer, M-MLV 5X 71505 1 ml $18.00 . , (2) 16 , 68 , 9326- and is used for and is used 260 2+ or Mn 2+ Methods Enzymol. J. Biol. Chem. , 50mM DTT. , eds.K. Maramorosch and H. , eds.K. Maramorosch 2 Methods In Molecular Biology . AMV Reverse Transcriptase is . AMV Reverse Transcriptase . Full-length copies of large . It consists of two polypeptide . It consists (3) (1) (1) . (4) 3' polymerase activity and the other an activity and the 3' polymerase , 250mM NaCl, 5mM DTT. , 250mM NaCl, 5mM 2 → Methods in Virology www.usbweb.com , 2365-2372. 22 , 143-184. 6 Innis, D. H. Gelfand and John J. Sni, (Academic Press, New York) p. 23. New York) Press, Innis, D. H. Gelfand and John J. Sni, (Academic 75-90. Biochemistry Humana Press, NJ, 73-93. Humana Press, 9335. 8.48. New York, Edition, Cold Spring Harbor, Manual, 3rd Koprowski Koprowski 4. (1990) eds. M. A. A Guide to Methods and Applications Protocols: PCR Concentration: 15 units/µl Storage Buffer: X-100, 50% 0.2% Triton (pH 7.2), 2mM DTT, 200mM potassium phosphate glycerol. Buffer (1 ml included, AMV RT Reaction 5X Functionally Tested PN 76218): (8.3), 40mM MgCl 250mM Tris-HCl References: 1. D. L. (1977) in Kacian, 3. H. (1983) D. M., Puskas, R. S., and Eschenfeldt, W. S. L., Wallace, Berger, 2. (2001) Molecular Cloning: A Laboratory J. and Russell, D. W. Sambrook, 3. and D’Alessio, J. M. (1993) G. F. Gerard, References: (1985) S. P. N. and Goff, 1. Roth, M. J., Tanese, first and second strand synthesis of complementary strand synthesis first and second mRNA from DNA (cDNA) can be conditions, high yields of full-length cDNA template. Under proper Transcriptase obtained with AMV Reverse mRNAs, >10 kb, may be synthesized. M-MLV Reverse Transcriptase has a Reverse Transcriptase mRNAs, >10 kb, may be synthesized. M-MLV in resulting Transcriptase much lower RNase H activity than AMV Reverse Reverse Transcriptase M-MLV high yields of full length cDNA. This makes allows for the RT very useful in the amplification of RNA. In addition, M-MLV at higher temperatures probes synthesis of DNA for cloning or hybridization chains, one of which contains a 5' of which contains chains, one RNase H activity. Reverse transcriptase requires Mg transcriptase requires Reverse RNase H activity. M-MLV Reverse Transcriptase M-MLV catalyzes the polymerization of DNA using Reverse Transcriptase M-MLV template DNA, RNA or RNA:DNA hybrids AMV Reverse Transcriptase Transcriptase AMV Reverse using of DNA catalyzes the polymerization Transcriptase AMV Reverse hybrids RNA or RNA:DNA template DNA, A major use for M-MLV Reverse Transcriptase is RT-PCR applications. See is RT-PCR Reverse Transcriptase A major use for M-MLV Kits, pages 10-15. USB RT-PCR Concentration: 200 units/µl Storage Buffer: 0.01% Nonidet 1mM DTT, 0.1mM EDTA, (pH 7.5), 0.1M NaCl, 20mM Tris-HCl P-40, 50% glycerol. Reaction Buffer (1 ml included, PN 71505): 5X M-MLV Functionally Tested MgCl (pH 8.3), 395mM KCl, 250mM Tris-HCl also useful in the amplification of RNA also useful in the amplification 2. H. M. and MacDonald, R. J. (1979) Goodman, M-MLV-RT AMV-RT ENZYMES 28 Analyze Kits Sequencing Phone: 1-800-321-9322 | Fax: 1-800-535-0898 sequencing reactions, resulting in very uniform band intensities. sequencing reactions, uniform resulting invery allows forequallyefficient incorporationofbothddNTPsanddNTPsincycle protocol whichrequires aslittlefmolamountsoftemplateDNA.Thekit Sequenase™Cycle Sequencing Kitfeatures anoptimized The Thermo Thermo Sequenase little as1fmol(orless)templateisrequired. Thisgreatlytermination. reduces theamountofradioactivelabelneeded. As Thus, onlysmallamountsoflabeledddNTPsare required forefficient with theadditionaladvantageofincorporatingddNTPsasreadily asdNTPs. asTaq SequenaseDNAPolymeraseisasthermostable Thermo Polymerase noBAFLs. background bandsandvirtually not visible.Gelresults are cleaner, more reliable andeasiertoread withfewer sequencing gel.Non-specificproducts remain unlabeled,andtherefore, are DNAsequencingfragmentsareproperly labeledandvisibleinthe terminated the sequencingreaction products atthe3’end.Thisensures thatonly This kitisuniqueinthatan misreading ofabase. prematurelyextensions aborting atrandompositions,whichcanleadto strong bandsinallfourlanes.Background bandscanbecausedbyprimer (ddNTP)andgive rise to bytheincorporationofaterminator terminated primerextensionproducts. Theseproductsaborted are notspecifically structure inG-C-richtemplateswhichcausestheaccumulationof secondary BAFLsresult fromand otherartifacts. thepolymerasepausingatregions of designed toeliminateBAFLs(BandsAcross FourLanes),background bands SequenaseRadiolabeledTerminatorThe Thermo CycleSequencingKitis Cycle SequencingKit Thermo SequenaseRadiolabeledTerminator r allowing gelelectrophoresis tobestoppedatanypointforanalysisandthen In allcases,thegelcanbescannedwithoutopeningglassplates,thus, (PN 79220).Nopurificationorpre-amplification isrequired. plasmids canbecarriedoutusingtheUSBYeast PlasmidIsolationKit inyeastliquidculture. plasmids Isolationofyeast sequencing from 0.2µ number ofsamplesinasingleworkingday. Thiskitcanalsobeusedfor rapidscreening andsequencingofalarge lambda plaques,permitting been optimizedforsequencingdirectly from singlebacterialcolonies,M13or detect fluorescently taggedsequencingproducts.The kitcomponentshave followed bystandard slabgelelectrophoresis andscanningofthegelto fluorescence scanners.Sequencingreactions involveonesteptermination designed tobeusedwithfluorescent dye-labeledprimersandhigh-resolution SequenaseDyePrimerManualCycleSequencingKitis The Thermo Cycle SequencingKit Thermo SequenaseDyePrimerManual included withthiskit. The method sequencing withouttheuseofcolumnsorotherlaboriousclean-upmethods. novel enzymaticclean-upmethodtopre-treat PCRproducts priorto The Sequenase™PCRProduct SequencingKitincludesSequenaseanda Sequenase PCRProductSequencingKit r r PCR product mixture, incubated at37°Candtheninactivated80°C.The the finalPCRproduct mixture. Bothenzymesare addedsimultaneouslytothe Phosphatase andExonucleaseItodegradeexcessdNTPsprimersin esumed iflongerread lengthsare needed. eady tobesequencedwithstandard SequenaseVersion 2.0Kitreagents, esult isacleanerPCRproduct, free from excessivenucleotidesandprimers, § involves theuseoftwohydrolytic enzymes,ShrimpAlkaline ª Cycle SequencingKit α - 33 P ddNTPradioactivelabelisincorporatedinto 0ratos$389.00 Includes 50 reactions 188403 Cycle SequencingKit Radiolabeled Terminator Thermo Sequenase $180.00 50 reactions 79260 Cycle SequencingKit Dye PrimerManual Thermo Sequenase 0ratos$157.00 Wi 50 reactions 79750 0 ecin $211.00 100 reactions 78500 Cycle SequencingKit Thermo Sequenaseª Wi $265.00 100 reactions 79760 0 ecin $238.00 100 reactions 70170 Sequencing Kit Sequenase PCRProduct thout thout 33 33 33 P P P ddNTPs ddNTPs ddNTPs Analyze 29 ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE . units/µl 20 ≥ 500 ml $36.00 500 ml5 lt $38.00 100 ml500 ml $48.00 $18.00 $23.00 400 ml400 ml $ 63.00 400 ml $ 63.00 $ 84.00 500 ml $ 38.00 torage Buffer: 20mM HEPES-KOH (pH 7.6), torage Buffer: MB Grade MB Grade MB Grade MB Grade MB Grade MB Grade TE Buffer, 50X Solution TE Buffer, ¥ 75834 100 ml $14.00 75896 Recombinant RNase Inhibitor, 100 ml¥S 50% glycerol with a 50mM KCl, 8mM DTT, concentration of 71571Acetate 3M Sodium $ 16.00 5,000 unitsSolution, pH 5.5 ¥ 75897 $125.00 100 ml 5X Solution TBE Buffer, ¥ 75891 1 lt 1X Solution TE Buffer, $16.00 ¥ 75893 10 x 1 ml $21.00 $14.00 Prod. No. 4.5, Equilibrated Phenol, pH Size77510 100 mlPhenol, pH 8.0, Equilibrated 75829 100 ml Phenol: Chloroform: Price Isoamyl Alcohol (25:24:1) $ 21.00 75831 100 mlAcetate 3M Potassium $ 21.00 Solution, pH 5.5 ¥ 75898 $ 26.00 100 ml2M Potassium Chloride Solution ¥ $ 16.00 2 ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE ULTRAPURE www.usbweb.com 1 kg $ 88.00 1 It $ 36.00 100 gm250 gm500 gm $108.00 $246.00 500 ml $389.00 5 gm25 gm $ 36.00 $ 50.00 $221.00 100 gm250 gm $324.00 100 gm $668.00 500 gm $200.00 $997.00 25 gm100 gm $ 48.00 $159.00 100 gm $286.00 B Grade, High Gel Strength 1,200gm/cm B Grade, Low MW Separation, <1,000 bp B Grade, Low MW Separation, >1,000 bp B Grade, High MW Separation, B Grade, pH 8.0 Low conductivity Free of contaminating nucleases. MB Grade, High Efficiency Separation 1,000 bp MB Grade, High Efficiency Ethidium Bromide Tablets Ethidium Bromide Tablets 7580875809 10 x 10 mgFormamide 10 x 100 mg¥ 75828 $ 28.00 500 gmGlycerol $ 38.00 ¥ 16374 500 ml $ 50.00 $ 22.00 EDTA, 0.5M Solution EDTA, ¥M 15694 100 mlEthidium Bromide 32813 1 gm $ 15.00 $ 12.00 Agarose ¥ 10132 25 gmAgarose ¥M 75817 $ 85.00 25 gm $ 33.00 Agarose, Low Melting/ Gelling Temperature ¥M 32829 25 gmAgarose, Low Melting/ Gelling Temperature ¥M 32830 $ 92.00 25 gm $108.00 Prod. No.Acridine Orange Size10390 5 gm Price $ 15.00 PCR Related Reagents 30 Analyze Reagents PCR Related Phone: 1-800-321-9322 | Fax: 1-800-535-0898 i.e., an authorized thermal cycler.i.e., anauthorizedthermal process iscovered by theup-front licensefee,eitherbypaymenttoPerkin-Elmer oraspurchased, ofthePCR cyclerwhose useintheautomatedperformance process inconjunctionwith athermal Purchase isaccompaniedbyalimitedlicensetouseitinthePolymerase Chain Reaction(PCR) †† § † Friendly are trademarksofUSBCorporation. Corporation. ‘FuelingInnovation’,‘Your inFuelingInnovation’,A-Taq-IT Partner andTested User ©2003 USBCorporation.USB,thelogodesign,andExoSAP-ITare registered trademarksofUSB and F. Hoffman-La RocheLtd. *The PolymeraseChainReaction(PCR) iscovered bypatentsownedRocheMolecularSystems T Nonidet isaregistered trademarkofShell. Sequenaseare trademarksofAmershamBiosciencesLimited. Sequenase andThermo process claimedinU.S.PatentNo.5,035,996. **Uracil DNAGlycosylase–Alicensemayberequired from LifeTechnologies, Inc.topracticethe and 5,674,716.PatentspendinginUSothercountries. or more ofthefollowingUSPatentNumbers:4,962,020;5,173,411;5,409,811; 5,498,523;5,614,365 SequenaseDNA Polymerase-ThisreagentThermo (kit)iscovered byorsuitableforuseunderone countries. 5,266,466; 5,409,811;5,498,523;5,639,608and5,674,716.Patentspendingin USandother US PatentNumbers:4,795,699;4,946,786;4,942,130;4,962,020;4,994,372; 5,145,776; 5,173,411; Sequenase DNAPolymerase-Thisreagent (kit)iscovered by orsuitableforuseunderonemore patents. license from AmershamBiosciencesCorp.underU.S.PatentNos.5,741,676and5,756,285related us [email protected]’llrushyouacopy! 2003-2004 USBcatalog.Ifyoudon’thaveacopy, pleasecallusore-mail For acompletelistingofagarosesandotherreagents,pleaseseeour Exonuclease I/Shrimp Alkaline Phosphatase – These products or portions thereofExonuclease I/ShrimpAlkalinePhosphatase–Theseproducts are orportions soldunder ExoSAP-IT iscovered byU.S.PatentNos.6,379,940 and 6,387,634. riton isaregistered trademarkofUnionCarbideChemicalsandPlasticsCompany. T aq DNAPolymerase–soldunderlicensing arrangementswithRocheMolecularSystems,Inc. rd o iePrice ¥ Size T Prod. No. 23 0 l$14.00 $14.00 100ml 22638 ¥ T $14.00 100ml 22639 ¥ T 100ml 22637 ris/HCl, 1MSolution,pH7.0 ris/HCl, 1MSolution,pH8.0 ris/HCl, 1MSolution,pH7.5 MB Grade MB Grade MB Grade t$32.00 $23.00 $32.00 1 lt $23.00 500 ml $32.00 1 lt $23.00 500 ml 1 lt 500 ml ULTRAPURE ULTRAPURE ULTRAPURE 08 0x1m $16.00 $28.00 10x1ml 70783 ¥ DEPC-Treated W 100ml 71788 $14.00 ¥ Endotoxin-Tested Price W 10x1ml 71786 ¥ Size W Prod. No. MB Grade MB Grade MB Grade ater, RNase-Free, ater, Nuclease-Free, ater, Nuclease-Free t$48.00 $34.00 $24.00 1 lt $54.00 500 ml 100 ml $86.00 $29.00 1 L $16.00 5 lt 1 lt 100 ml ULTRAPURE ULTRAPURE ULTRAPURE USB Corporation

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