Protocol for EZ-Tn5™ Insertion Kit
Transcript of Protocol for EZ-Tn5™ Insertion Kit
Connect with Epicentre on our blog (epicentral.blogspot.com), Facebook (facebook.com/EpicentreBio), and Twitter (@EpicentreBio).
EZ-Tn5™ <KAN-2> Insertion KitCat. Nos. EZI982K, EZI96K2
www.epicentre.com Lit.#134•8/2012 1 EPILIT134 Rev. A
2 www.epicentre.com
EZ-Tn5™ Transposon Tools
1. IntroductionTransposons are mobile DNA sequences found in the genomes of prokaryotes and eukaryotes. Transposon tagging has long been recognized as a powerful research tool for randomly distributing primer binding sites, creating gene “knockouts”, and introducing a physical tag or a genetic tag into large target DNAs. One frequently used transposition system is the Tn5 system isolated from gram-negative bacteria.Though a naturally occurring transposition system, the Tn5 system can be readily adapted for routine use in research laboratories for the following reasons:1) Tn5 transposase is a small, single subunit enzyme that has been cloned and purified
to high specific activity.2) Tn5transposasecarriesouttranspositionwithouttheneedforhostcellfactors.3) Tn5 transposon insertions into target DNA are highly random.4) Tn5 transposition proceeds by a simple “cut and paste” process. Although the
chemistry is unique, the result is similar to using a restriction endonuclease, with random sequence specifi city, accompanied by a DNA ligase activity.
5) Tn5 transposase will transpose any DNA sequence contained between its short 19 basepair Mo saic End (ME) Tn5 transposase recognition sequences.
In1998GoryshinandReznikoff 1 demonstrated that a fully functional Tn5 transposition system could be reconstituted in vitro. Additionally, the transposition efficiency of thissystemhasbeenincreasedmorethan1,000-foldcomparedtowild-typeTn5byintroducing mutations in the transposase gene and in the 19-bp Tn5 ME transposase recognition sequence. Epicentre’s EZ-Tn5 Transposon Tools (kits and reagents) are based onthehyperactiveTn5transpositionsystemdevelopedbyGoryshinandReznikoff.
2. Kit ContentsCat. # Concentration Quantity
Reagentsincludedinthekitaresufficientfor10in vitro transposon insertion reactions.
EZ-Tn5™ Transposase:10 U @1 U/µl 10µlin50%glycerolcontaining50 mMTris-HCl(pH 7.5),0.1 MNaCl,0.1 mMEDTA,1 mMdithiothreitol,and0.1%Triton®X-100.
EZ-Tn5™ <KAN-2> Transposon:[email protected] pmol/µl(0.081 µg/µl) 10µlinTEBuffer(10 mMTris-HCl[pH7.5]and1 mMEDTA).
EZ-Tn5™ 10X Reaction Buffer: 100µl0.50 MTris-acetate(pH7.5),1.5 Mpotassiumacetate,100 mMmagnesiumacetate,and40 mMspermidine.
EZ-Tn5™ 10X Stop Solution: 100µl1%SDSsolution.
KAN-2 FP-1 Forward Primer:1nmol @50µM 20µlinTEBuffer(10 mMTris-HCl[pH7.5]and1 mMEDTA).
KAN-2 RP-1 Reverse Primer:1nmol @50µM 20µlinTEBuffer(10 mMTris-HCl[pH7.5]and1 mMEDTA).
pUC19/3.4 Control Target DNA:1µg @0.1 µg/µl 10µlA 3.4-kb Hpa II fragment of bacteriophage DNA cloned into the Acc IsiteofpUC19, inTEBuffer(10 mMTris-HCl[pH7.5]and1 mMEDTA).
Sterile Water: 1 ml
[email protected]•(800)284-8474 3
EZ-Tn5™ Transposon Tools
3. Related ProductsThe following products are also available:– TransforMax™EC100™ElectrocompetentE. coli •Transformationefficiency>109cfu/µgDNA.– ColonyFast-Screen™Kits– MasterPure™DNAPurificationKits– EZ-Tn5™TnpTransposome™Kits– EZ-Tn5™ Transposase– EZ-Tn5™ Transposons– EZ-Tn5™InsertionKits
How the EZ-Tn5 <KAN-2> Insertion Kit Works.TheEZ-Tn5<KAN-2>InsertionKitcanbeusedtorandomlyinsertprimerbindingsitesand a kana mycin resistance selection marker into target DNA in vitro.Asingle2-hourin vitroreactionrandomlyinsertsthe<KAN-2>TransposonintothetargetDNA.Usean aliquot of the reaction to transform E. colisuchasEpicentre’sTransforMax™EC100™Electrocompetent E. coli strain and select on kanamycin plates. Only those clones harboringDNAcontainingthe<KAN-2>Transposonwillgrow.TheEZ-Tn5<KAN-2>InsertionKitcanbeusedfor:• FastersequencingoflargeDNAmolecules,ascomparedtoprimerwalking,random
subcloning, or generating nested deletions with exonuclease III and mung bean nuclease.
• Makinginsertionmutantsorgene“knockouts”in vitro.• IntroducingakanamycinresistanceselectionmarkerintoanyDNA.
Fig.1describesthestepsinvolvedwhenusingtheEZ-Tn5<KAN-2>InsertionKit.Theprocess can be summarized as follows:
Preparation• Prepare0.2 µgofrecombinantDNAfortheEZ-Tn5<KAN-2>insertionreaction.Day 1• Performthe2-hourin vitroEZ-Tn5<KAN-2>insertionreaction.• TransformcompetentrecA— E. coliwith1 µlofthereactionmix.• Selectforkanamycin-resistanttransposoninsertionclonesonkanamycinplates
overnight.Day 2• PrepareDNAfromkanamycin-resistantcolonies.• (Optional)MaptheEZ-Tn5<KAN-2>Transposoninsertionsites.• (Optional)DNAsequencechosenclonesbidirectionallyusingtheunlabeledforward
and reverse transposon-specific primers supplied in the kit.
4 www.epicentre.com
EZ-Tn5™ Transposon Tools
1. Incubate 37oC; 2 hrs2. Transform E. coli3. Select KanR clones
Sequence bidirectionally from primer binding sites ( )
4. Prepare plasmid DNA
EZ-Tn5<KAN-2>
TransposonEZ-Tn5
Transposase
KANRTargetDNA
Figure 1. EZ-Tn5 <KAN-2> Transposon Insertion Protocol.
[email protected]•(800)284-8474 5
EZ-Tn5™ Transposon Tools
4. MaterialsStorage
StoreEZ-Tn5InsertionKitsonlyat–20°Cinafreezerwithoutadefrostcycle.Theenzymesolution will not freeze. Other component solutions will freeze. After thawing, be sure to mix the contents of each tube thoroughly before using. After use, return all of the kit componentsto–20°Cforstorage.
Performance Specifications and Quality Control
TheEZ-Tn5<KAN-2>InsertionKitisfunction-testedbyperformanceofthekit’sin vitro control reac tion followed by electroporation into a recA— E. coli host strain having a transformationefficiencyof>109 cfu/µgDNA.Transpositionfrequency,definedastheratioofthenumberofKanR clones divided by the number of transformants resistant to theantibioticmarkerofthetargetvector,(KanRcolonies/AmpR colonies; for the control DNA)mustbe>0.5%(commonlyat10%)andtranspositionefficiencymustbe>106KanR colonies/mgtargetDNA.Primersarefunction-testedinaDNAcyclesequencingreactionusingtheSequiThermEXCEL™IIDNASequencingKitandinaPCRreactionusingaplasmidcontaininganEZ-Tn5<KAN-2>Transposonastemplate.AllcomponentsoftheEZ-Tn5<KAN-2>InsertionKitarefreeofdetectableDNaseandRNaseactivitiesasjudgedby agarose gel electro phoresis following over-digestion assays, with the exception of the inherent endonucleolytic function of the EZ-Tn5 Transposase.
5. Transposon Insertion ReactionTarget DNA Preparation
The target DNA must not contain a kanamycin resistance gene. The transposon insertion reaction is not significantly inhibited by high levels of RNA contamination in target DNA preparations.However,ifthetargetDNAisheavilycontaminatedwithchromosomalDNA, which is a direct competitor for tar get transposition, the number of clones will be greatly reduced.
Plasmid and cosmid clones can be purified by standard minilysate procedures and used as target DNA in the insertion reaction without further clean-up. Low copy-number vectors, for example BAC or fosmid clones, are often contaminated with a higher molar proportion of E. coli chromosomal DNA thus reducing the transposon insertion frequency. Therefore, BAC and fosmid DNA should be puri fied, to remove the chromosomal DNA prior to the insertion reaction.
In Vitro Transposon Insertion Reaction
Reaction conditions have been optimized to maximize the efficiency of the EZ-Tn5 Transposon inser tion while minimizing multiple insertion events. Be sure to calculate the moles of target DNA used in the reaction and add an equimolar amount of the EZ-Tn5 <KAN-2> Transposon.Ifnecessary,dilutetheEZ-Tn5<KAN-2>TransposonwithTEbuffer(10 mMTris-HCl[pH 7.5],1 mMEDTA).
6 www.epicentre.com
EZ-Tn5™ Transposon Tools
1. Prepare the transposon insertion reaction mixture by adding in the following order:
1 µl EZ-Tn510XReactionBuffer 0.2 µgtargetDNA* x µl molarequivalentEZ-Tn5<KAN-2>Transposon x µl sterilewatertoareactionvolumeof9 µl 1 µl EZ-Tn5Transposase
10 µl Totalreactionvolume
*CalculationofµmoletargetDNA:
µmoltargetDNA=µgtargetDNA/[(#basepairsintargetDNA)x660]
Forexample:0.2µgofcontrolpUC19/3.4DNAwhichis6,100bp
=0.2µg/[6,100 bpx660]=0.05x10-6µmol=0.05 pmol
2. Incubatethereactionmixturefor2hoursat37°C.
3. Stopthereactionbyadding1 µlEZ-Tn510XStopSolution. Mixandheatfor10minutesat70°C. Proceed to section 4, Transformation and Recovery or store the reaction mixture at –20°C.
6. Selection of Transposon Insertion ClonesTransformation and Recovery
The number of EZ-Tn5 Transposon insertion clones obtained per reaction depends on, among other factors, the transformation efficiency of the competent cells used. The greater the transformation effi ciency of the competent cells, the greater the number of insertion clones obtained. We recommend using electrocompetent or chemically competent recA— E. coliwithatransformationefficiencyof>108 cfu/µgofDNA.ArecA— strain of E. coli is recommended to eliminate the possibility of generat ing multimeric forms of the vector. Finally, the host strain used must not have a kanamycin resist ance markerwhenusedwiththeEZ-Tn5<KAN-2>Transposon.Epicentre’sTransforMaxEC100Electrocompetent E. coli(availableseparately)haveatransformationefficiencyof>1x109cfu/µgandareidealforthisapplication.
1) Using1 µloftheinsertionreactionmixture,transformrecA— E. coli. If electrocompetent cells are used, perform electroporation according to the equipmentmanufacturer’srecommendedconditions.Useof>1 µlofthetransposoninsertion reaction for transformation may result in arcing. The unused portion of the transposoninsertionreactioncanbestoredat–20°Cforfutureuse.
2) RecovertheelectroporatedcellsbyaddingSOCmediumtotheelectroporationcuvette to 1 ml final volume immediately after electroporation. Pipette the medium/cellsgentlytomix.Transfertoatubeandincubateona37°Cshakerfor30-60minutestofacilitatecelloutgrowth.
[email protected]•(800)284-8474 7
EZ-Tn5™ Transposon Tools
Plating and Selecting Transformants
EZ-Tn5<KAN-2>Transposoninsertionclonesareselectedonkanamycin-containingplates,however,thetransposonwillalsoconferresistancetoneomycinandG418inE. coli.
1) Iftransformationwasdoneusingcellswithanefficiencyof>5x108 cfu/µgDNA,itmaybenecessarytodilutethecells1:10or1:100priortoplating.Plateportions(e.g.,100 µl)ofcellsontoLBplatescontaining50 µg/mlkanamycin.Storetheunusedportionoftherecoveredcellsat+4°Cforupto2daysintheeventadditionalplates need to be prepared.
2) (Optional)Todeterminethetransposoninsertionefficiency,plateidenticaldilutionsand dilution aliquots of the transformation reaction on a second plate containing an antibioticspecificforselectingtargetDNA(e.g.,100 µg/mlampicillinforthecontrolDNA).ThetranspositionfrequencyisgivenbytheratioofKanR/AmpR clones for the control DNA.
3) Growplatesovernightat37°C.Assumingatransposoninsertionefficiencyof1%anduseofhighpuritytargetDNA(i.e.,littleornochromosomalDNAcontamination),oneshouldsee100-500KanR clones per plate. If too few (or too many) colonies appear, replate the transformed cells at a lower (or higher) dilution.
TypicalresultsobtainedwiththeEZ-Tn5<KAN-2>InsertionKitare:• Transposoninsertionfrequency=0.5-20%• TranspositionclonesperµgtargetDNA=1x105-1x108
• Transpositionclonesper10 µltransposoninsertionreaction=1x104-1x107
TheactualnumberofEZ-Tn5<KAN-2>insertionclonesobtainedwillvarydependingonfactors such as target DNA size and the transformation efficiency of the competent cells used to recover the trans poson insertion clones.
7. DNA Sequencing of Transposon Insertion ClonesTransposon Insertion Mapping (optional)
EZ-Tn5 Transposon insertion clones can be sequenced bidirectionally using the unlabeled forward and reverse transposon-specific primers provided in the kit. The insertion site of each clone can also be mapped prior to sequencing, depending on the cost-effectivenessandexperienceoftheuser.
EZ-Tn5<KAN-2>TransposoninsertionsitescanbemappedbysizeanalysisofPCRproducts using colony minilysate DNA as a template. To map the insertion sites, use the KAN-2FP-1orKAN-2RP-1primersprovidedwiththekitandavector-specificflankingprimers (not provided).
Alternatively,insertionsitescanbemappedbyrestrictionendonucleasedigest(s).UsethenucleotidesequenceandrestrictioninformationoftheEZ-Tn5<KAN-2>Transposonprovided in the Appen dix for reference.
8 www.epicentre.com
EZ-Tn5™ Transposon Tools
Primer Considerations
TheKAN-2FP-1ForwardandKAN-2RP-1ReversePrimerssuppliedwiththekithavebeencon structed to minimize homology to commonly used cloning vectors. However, the sequence of each primer should be compared to that of the user’s specific cloning vector to ensure minimal se quence homology to the vector. The sequence and theoretical melting temperatures for each pri mer are presented in the Appendix.
Note: Occasionally a clone will yield the sequence of the cloning vector. This occurs when the EZ-Tn5 Transposon randomly inserts into a non-essential region of the vector rather than into the DNA insert. The frequency of this occurrence is dependent on the size of the DNA insert relative to the size of non-essential regions of vector. The larger the DNA insert, the less frequently an insertion will occur into the vector.
Target Site Duplication
EZ-Tn5 Transposase-catalyzed transposon insertion results in the generation of a 9-bp targetsitesequenceduplicationwhereonecopyimmediatelyflankseachsideoftheinserted transposon. This is important to consider when assembling the nucleotide sequence of a recombinant clone insert. The process of transposon insertion site duplicationisdepictedinFig. 2.
Distinguishing Transposon Sequence from Insert Sequence
SincetheprimersprovidedintheEZ-Tn5<KAN-2>InsertionKitannealtoaregionnearthe ends of the transposon, the first sequence data obtained from each sequencing reactionisthatofEZ-Tn5<KAN-2>TransposonDNA.Thesequenceofthe19-bpEZ-Tn5Transposaserecognitionsequence(ME)foundatthejunctionofthetransposonandthe target clone insert DNA (present in all insertion clones), is a useful landmark to distinguish vector sequence from target clone insert sequence (see also Fig. 3).
8. TroubleshootingArcing during transformation by electroporation
1) Excessive salt in target DNA preparation.Uselesssampleforelectroporation.Ethanolprecipitateand70%ethanolwash,ordropdialyzeDNA.
Arcing inevitably results in failed transformation. Discard the electroporation reactionandtryagainwith0.5 µlofthetranspositionreaction.WiththecontrolDNA,noarcingisobservedwhenupto2 µloftranspositionreactionisusedforelectroporationof50 µlofelectrocompetentrecA— E. coliina0.2-cmwidthelectroporationcuvetteandusinganEppendorfMultiporatorat2500 V.Avoltagegradientofapproximately12,500 V/cmisfairlystandardforE. coli.
No, or few transposition clones on selective plates1) Transformation reaction was unsuccessful; low competence. Test by plating
outgrowth using drug resistance marker on target DNA to distinguish between transformation or transposon inser tion failure.
[email protected]•(800)284-8474 9
EZ-Tn5™ Transposon Tools
9 b.p. Staggered Cut
Target DNA5'3'
A B C D E F G H I
A B C D E F G H I ' ''''''''
9 b.p. Gaps5'3' A B C D E F G H I ' ''''''''
A B C D E F G H I
MEME
+EZ-Tn5™ TRANSPOSASE
EZ-Tn5™TRANSPOSON
MEME
E. coli DNA Repair after Transformation
5'3'
A B C D E F G H I
A B C D E F G H I ' ''''''''A B C D E F G H I
A B C D E F G H I ' ''''''''
MEME9 b.p.Direct RepeatTarget Site Duplication
Transesterification Reaction
Random Transposon Insertion
5'
3' GACAGAGAATATGTGTAGACTGTCTCTTATACACATCT AGATGTGTATAAGAGACAG
TCTACACATATTCTCTGTCEZ-Tn5™ TRANSPOSON
19 bp Mosaic End Sequence19 bp Mosaic End Sequence
ForwardPrimer
ReversePrimer
9 b.p. Staggered Cut
Target DNA5'3'
A B C D E F G H I
A B C D E F G H I ' ''''''''
9 b.p. Gaps5'3' A B C D E F G H I ' ''''''''
A B C D E F G H I
MEME
+EZ-Tn5™ TRANSPOSASE
EZ-Tn5™TRANSPOSON
MEME
E. coli DNA Repair after Transformation
5'3'
A B C D E F G H I
A B C D E F G H I ' ''''''''A B C D E F G H I
A B C D E F G H I ' ''''''''
MEME9 b.p.Direct RepeatTarget Site Duplication
Transesterification Reaction
Random Transposon Insertion
5'
3' GACAGAGAATATGTGTAGACTGTCTCTTATACACATCT AGATGTGTATAAGAGACAG
TCTACACATATTCTCTGTCEZ-Tn5™ TRANSPOSON
19 bp Mosaic End Sequence19 bp Mosaic End Sequence
ForwardPrimer
ReversePrimer
Figure 2. EZ-Tn5 Transposase Insertion Site Duplication Process.
Figure 3. EZ-Tn5 Transposon Insertion Site Junction.
Ifcompetentcellshaveatransformationefficiency<108 cfu/µgDNA,onemaynot obtain suffi cient clones on a plate. For example, transforming into cells with transformationefficiency<105 cfu/µgDNAresultsinasfewas2insertionclonesonaplate.Usecellswithatransformationefficiency>108 cfu/µgDNA.
2) Transposon insertion reaction was unsuccessful. Inhibitor contamination in target DNA. Pur ify target DNA further. Perform procedure with control plasmid provided with kit to assure system components are functional.
10 www.epicentre.com
EZ-Tn5™ Transposon Tools
DNA sequencing results are ambiguous
1) Two or more transposon insertions into target clone. Discard the clone. Choose other clone(s) to sequence.
The protocol was designed to minimize multiple transposon insertion events. Even so,about1%ofthetranspositionclonesmaycontain>1transposon.Onecanverifysingle insertion clones by agarose gel electrophoresis of colony minilysates prior to sequencing.Asingleinsertionshouldbeabout1.2 kblargerthantheparentaltargetDNA.Adoubleinsertionwillincreasethesizeby2.4 kbandalsoresultin“doublesequence”.
2) The sequencing primer used has significant homology to the cloning vector or to the DNA being sequenced. Check homology of primer against the vector. Alter primer annealing condi tions or synthesize a new primer with less homology.
3) Components of the DNA sequencing kit and/or of the electrophoresis step are compromised. Reverifytheintegrityofthecomponentsofthekitand/orelectrophoresisstepwithappropriatecontrols.Useanewkitand/ornewreagents.
Confluent plates following transformation1) Target DNA or host cells have the same selective marker (antibiotic resistance)
as the transposon used.Useadifferenthostfortransformationandretransformwith a portion of your remaining reaction.
SomehostcellscarryKanR transposons. Confirm that the genotype of the host strain chosenforthetransformationisnotKanR.
9. Appendix
Primer DataKAN-2 FP-1 Forward Primer
5′ - ACCTACAACAAAGCTCTCATCAACC - 3′
Length:25nucleotidesG+C content: 11
Molecular Weight:7,484daltonsTemperatures of Dissociation & Melting:Td: 68°C (nearestneighbormethod)
Tm: 73°C (%G+Cmethod)
Tm: 72°C ([2(A+T)+4(G+C)]method)
Tm: 63°C ((81.5+16.6(log[Na+]))+([41(#G+C)-500]/length)method) where[Na+]=0.1M
[email protected]•(800)284-8474 11
EZ-Tn5™ Transposon Tools
Figure 4. EZ-Tn5 <KAN-2> Transposon.
EZ-Tn5 <KAN-2> Transposon Schematic Map
EZ-Tn5™ <KAN-2> Transposon(1,221 bp)
KAN-2 FP-1 Forward PrimerKAN-2 RP-1 Reverse PrimerME = Mosaic End
5' ACCTACAACAAAGCTCTCATCAACC 3'5' GCAATGTAACATCAGAGATTTTGAG 3'5' AGATGTGTATAAGAGACAG 3'
KANR
BspH I 87Xho I 167
Nru I 226
Nci I 442Ssp I 494
Pvu I 569Hae I 656
PflM I 832
Ava II 1122BamH I 1159
Xba I 1165Sal I 1171
Acc I 1172Hinc II 1173
Pst I 1181Sph I 1187
Hind III 1189
ME MEATG138
TAA953 KAN-2 FP-1
1127-1151KAN-2 RP-1
43-67
Note: Not all restriction enzymes that cut only once are indicated above.
Primers are not drawn to scale.See the following pages for further information.
1 100 200 300 500 700 900 1221400 600 800 1000 1100
KAN-2 RP-1 Reverse Primer
5′ - GCAATGTAACATCAGAGATTTTGAG - 3′
Length:25nucleotidesG+C content: 9Molecular Weight:7,705daltonsTemperatures of Dissociation & Melting:Td: 65°C (nearestneighbormethod)
Tm: 69°C (%G+Cmethod)
Tm: 68°C ([2(A+T)+4(G+C)]method)
Tm: 60°C ((81.5+16.6(log[Na+]))+([41(#G+C)-500]/length)method) where[Na+]=0.1M
12 www.epicentre.com
EZ-Tn5™ Transposon Tools
EZ-Tn5 <KAN-2> Transposon Restriction Data
Restriction Enzymes that cut the EZ-Tn5 <KAN-2> Transposon one to three times:
Enzyme Sites LocationAccI 1 1172AciI 1 174AluI 2 1139,1191ApoI 2 182,366AsiSI 1 569AseI 1 768AvaI 1 167AvaII 1 1122BamHI 1 1159BanII 1 224Bfa I 1 1166BfrBI 2 417,683BfuAI 1 1184Bpu10I 1 586BsaWI 1 704BsiE I 1 569BsmI 2 453,530BsmBI 1 585Bsp1286I 1 224BspDI 2 29,260BspHI 1 87BspMI 1 1184BsrI 3 360,984,1124BsrD I 1 61BsrFI 1 523BssKI 3 440,457,814BstF5I 2 200,826BstNI 2 459,816BstUI 3 176,226,571BstYI 2 818,1159BtgI 2 1089,1150BtsI 2 430,517ClaI 2 29,260DdeI 2 586,1041DsaI 2 1089,1150EarI 1 382EcoNI 1 481FauI 1 1148
Enzyme Sites Location Fnu4HI 2 174,1013HaeI 1 656HaeIII 2 173,656HincII 1 1173HindIII 1 1189HpaII 3 442,524,705HpyCH4IV 1 159Mae II 1 159MboII 3 369,480,1058MlyI 2 802,1178MspI 3 443,525,706MwoI 3 281,313,527NciI 1 442NlaIV 1 1161NruI 1 226NsiI 2 419,685NspI 1 1187PaeR7I 1 167PflMI 1 832PleI 2 801,1177PspGI 2 457,814PstI 1 1181Pvu I 1 569RsaI 1 404SalI 1 1171Sau96I 1 1122SbfI 1 1181ScrFI 3 442,459,816SfcI 1 1177SmlI 1 167SphI 1 1187SspI 1 494TliI 1 167Tsp45I 1 716TspRI 3 442,517,989XbaI 1 1165XhoI 1 167
[email protected]•(800)284-8474 13
EZ-Tn5™ Transposon Tools
Restriction Enzymes that cut the EZ-Tn5 <KAN-2> Transposon four or more times:
Alw IBsaJ IBsl IBsmA ICac8I
CviJ IDpn IHhaIHinfIHinPI
HphIHpy188IHpyCH4IIIHpyCH4VMae III
Mbo IMnl IMse INla IIISau3AI
SfaNITaq ITfi ITsp4C ITsp509I
Aat IIAcc65 IAcl IAfe IAflIIAflIIIAge IAhd IAle IAlwN IApa IApaB IApaL IAsc IAvr IIBan IBbs IBbvC IBciVIBcl IBgl IBgl II
Blp IBme1580IBmgB IBmr IBsa IBsaA IBsaB IBsaHIBseY IBsiHKAIBsiW IBspE IBspLU11IBsrB IBsrGIBssHIIBssSIBstAP IBstB IBstE IIBstXIBstZ17I
Bsu36 IDra IDra IIIDrd IEae IEag IEco47IIIEcoO109IEcoR IEcoRVFse IFsp IGdiIIHaeIIHpaIHpy99IKpnIMfe IMlu IMsc IMsl IMspA1 I
Nae INar INco INde INgoMIVNhe INot IPac IPci IPflFIPme IPml IPpuM IPshA IPsi IPspOM IPvu IIRsr IISacISacIISanDISapI
ScaISexAISfiISfoISgrAISimISmaISnaBISpeISrfISse8647IStuIStyISwaITat ITse ITth111 IXcmIXmaIXmnI
Restriction Enzymes that do not cut the EZ-Tn5 <KAN-2> Transposon:
14 www.epicentre.com
EZ-Tn5™ Transposon Tools
EZ-Tn5 <KAN-2> Transposon Sequence
EZ-Tn5™ <KAN-2> Transposon 1,221 bp.
1 CTGTCTCTTA TACACATCTC AACCATCATC GATGAATTGT GTCTCAAAAT
51 CTCTGATGTT ACATTGCACA AGATAAAAAT ATATCATCAT GAACAATAAA
101 ACTGTCTGCT TACATAAACA GTAATACAAG GGGTGTTATG AGCCATATTC
151 AACGGGAAAC GTCTTGCTCG AGGCCGCGAT TAAATTCCAA CATGGATGCT
201 GATTTATATG GGTATAAATG GGCTCGCGAT AATGTCGGGC AATCAGGTGC
251 GACAATCTAT CGATTGTATG GGAAGCCCGA TGCGCCAGAG TTGTTTCTGA
301 AACATGGCAA AGGTAGCGTT GCCAATGATG TTACAGATGA GATGGTCAGA
351 CTAAACTGGC TGACGGAATT TATGCCTCTT CCGACCATCA AGCATTTTAT
401 CCGTACTCCT GATGATGCAT GGTTACTCAC CACTGCGATC CCCGGAAAAA
451 CAGCATTCCA GGTATTAGAA GAATATCCTG ATTCAGGTGA AAATATTGTT
501 GATGCGCTGG CAGTGTTCCT GCGCCGGTTG CATTCGATTC CTGTTTGTAA
551 TTGTCCTTTT AACAGCGATC GCGTATTTCG TCTCGCTCAG GCGCAATCAC
601 GAATGAATAA CGGTTTGGTT GATGCGAGTG ATTTTGATGA CGAGCGTAAT
651 GGCTGGCCTG TTGAACAAGT CTGGAAAGAA ATGCATAAAC TTTTGCCATT
701 CTCACCGGAT TCAGTCGTCA CTCATGGTGA TTTCTCACTT GATAACCTTA
751 TTTTTGACGA GGGGAAATTA ATAGGTTGTA TTGATGTTGG ACGAGTCGGA
801 ATCGCAGACC GATACCAGGA TCTTGCCATC CTATGGAACT GCCTCGGTGA
851 GTTTTCTCCT TCATTACAGA AACGGCTTTT TCAAAAATAT GGTATTGATA
901 ATCCTGATAT GAATAAATTG CAGTTTCATT TGATGCTCGA TGAGTTTTTC
951 TAATCAGAAT TGGTTAATTG GTTGTAACAC TGGCAGAGCA TTACGCTGAC
1001TTGACGGGAC GGCGGCTTTG TTGAATAAAT CGAACTTTTG CTGAGTTGAA
1051GGATCAGATC ACGCATCTTC CCGACAACGC AGACCGTTCC GTGGCAAAGC
1101AAAAGTTCAA AATCACCAAC TGGTCCACCT ACAACAAAGC TCTCATCAAC
1151CGTGGCGGGG ATCCTCTAGA GTCGACCTGC AGGCATGCAA GCTTCAGGGT
1201TGAGATGTGT ATAAGAGACA G
ThetransposonsequencecanbedownloadedattheURL:http://www.epicentre.com/sequences
[email protected]•(800)284-8474 15
EZ-Tn5™ Transposon Tools
10. ReferencesCited:
1. Goryshin,I.Y.andReznikoff,W.S.(1998)J. Biol. Chem. 273,7367.
Development of the hyperactive EZ-Tn5 (Tn5) in vitro transposition system:
1. Goryshin,I.Y.andReznikoff,W.S.(1998)J. Biol. Chem. 273,7367.2. Zhou,M.et al.,(1998)J. Mol. Biol. 276, 913.
3. Zhou,M.andReznikoff,W.S.(1997)J. Mol. Biol. 271,362.4. Mahnke-Braam,I.A.andReznikoff,W.S.(1998)J. Biol. Chem. 273,10908.
These products are intended for research use only.
EZ-Tn5™ Transposon Tools for in vitro transposon insertion are covered by U.S. Patent Nos. 5,925,545; 5,948,622; 5,965,443, and 6,437,109; European Patent No. 0927258, and other pat ents issued or pending, exclusively licensed or assigned to Epicentre. These products are accom-panied by a limited non-exclusive license for the purchaser to use the purchased product(s) solely for in vitro transposon insertion for life science research. Purchase of these products does not grant rights to: (1) offer products, components of products, or any derivatives thereof for resale; or (2) to distribute or transfer the products, components of products, or any derivatives thereof to third parties. Contact Epicentre for information on licenses for uses other than life science research.
Triton is a registered trademark of Rohm & Haas, Philadelphia, Pennsylvania.
EC100, EZ-Tn5, Fast-Screen, TransforMax, MasterPure, SequiTherm EXCEL, and Transposome are trademarks of Epicentre, Madison, Wisconsin.
Visitourtechnicalblog:epicentral.blogspot.com