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pENTR™ Directional TOPO® Cloning Kits

1. 0 5 mM EDTA pH 8 Reagent Composition Amount S O C Medium 2 Tryptone 6 ml may be stored at room 0 5 Yeast Extract temperature or 4 C 10 mM NaCl 25mMKCI 10 mM MgCb 10 mM MgSO 20 mM glucose TOP10 or Mach1 T1 21x50 ul cells pUC19 Control DNA 10 pg ul in 5 mM Tris HCl 50 ul TOP10 F mcrA A mrr hsdRMS mcrBC 80lacZAM15 AlacX74 rec A1 araD139 A ara leu 7697 galU galK rpsL Str end A1 nupG Mach1 T18 F 80lacZAM15 AlacX74 hsdR rc m ArecA1398 end A1 ton A confers resistance to phage T1 The parental strain of Mach1 T18 EF coli is the non K 12 wild type W strain ATCC 9637 S A Waksman Although the parental strain is generally classified as Biosafety Level 1 BL 1 we recommend that you consult the safety department of your institution to verify the Biosafety Level ix Accessory Products Introduction Additional Products The products listed in this section may be used with the pENTR Directional TOPO Cloning Kits For more information refer to www invitrogen com or call Technical Service see page 35 Many of the reagents supplied in the pENTR Directional TOPO Cloning Kits and other reagents suitable for use with the kits are available separately from Invitrogen Ordering information for these reagents is provided below Item Quantity Catalog no One Shot TOP10 Chemically Competent
2. Technical Service Web Resources Visit the Invitrogen Web site at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical service contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our Web page www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 1600 Faraday Avenue LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech serviceQinvitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com Material Data Safety Sheets MSDSs Limited Warranty MSDSs are available on our Web site at www invitrogen com On the home page click on Technical Resources and follow instructions on the page to download the MSDS for your product Invitrogen is committed to providing our customers with high quality goods and services Our goal is
3. 10 reactions C4040 10 Bion 20 reactions C4040 03 One Shot TOP10 Electrocompetent E coli 10 reactions C4040 50 One Shot Mach1 T1 Chemically 20 reactions C8620 03 Competent E coli M13 Forward 20 Primer 2 ug 407 pmoles N520 02 M13 Reverse Primer 2 ug 385 pmoles N530 02 Kanamycin Sulfate 1g 11815 016 LB Broth 500 ml 10855 021 LB Agar 500g 22700 025 PureLink HQ Mini Plasmid Purification 100 reactions K2100 01 Kit Gateway LR Clonase II Enzyme Mix 20 reactions 11791 020 100 reactions 11791 100 Gateway LR Clonase Plus Enzyme Mix 20 reactions 12538 013 MultiSite Gateway Three Fragment Vector 1 kit 12537 023 Construction Kit AcTEV Protease 1 000 units 12575 015 10 000 units 12575 023 Overview Introduction The Gateway Technology Introduction The pENTR Directional TOPO Cloning Kits utilize a highly efficient 5 minute cloning strategy TOPO Cloning to directionally clone a blunt end PCR product into a vector for entry into the Gateway System or the MultiSite Gateway System available from Invitrogen Blunt end PCR products clone directionally at greater than 90 efficiency with no ligase post PCR procedures or restriction enzymes required A choice of pENTR Directional TOPO vectors is available for optimal expression of your PCR product after recombination with the Gateway destination vector of interest see table below Vector Benefit pENTR D TOPO For
4. General guidelines are provided below For most applications we recommend performing the LR recombination reaction or the MultiSite Gateway LR recombination reaction using a e Supercoiled entry clone s and e Supercoiled destination vector To catalyze the LR recombination reaction we recommend using Gateway LR Clonase II Enzyme Mix see page x for ordering information The LR Clonase II enzyme mix combines the proprietary enzyme formulation and 5X LR Reaction Buffer previously supplied by Invitrogen as separate components in LR Clonase enzyme mix Catalog no 11791 019 into an optimized single tube format to allow easier set up of the LR recombination reaction Follow the instructions included with the product to perform the LR recombination reaction Note You may perform the LR recombination reaction using LR Clonase enzyme mix if desired A large selection of Gateway destination vectors is available from Invitrogen to facilitate expression of your gene of interest in virtually any protein expression system For more information about the vectors available see our Web site www invitrogen com or call Technical Service see page 35 Manuals supporting all of the destination vectors are available for downloading from our Web site or by contacting Technical Service Once you have performed the LR recombination reaction or the MultiSite Gateway LR recombination reaction you will transform the reaction mixture
5. Step 2 page 12 into a sterile microcentrifuge tube containing 50 ul of electrocompetent E coli and mix gently Do not mix by pipetting up and down Avoid formation of bubbles Transfer the cells to a 0 1 cm cuvette 2 Electroporate your samples using your own protocol and your electroporator Note If you have problems with arcing see the next page 3 Immediately add 250 ul of room temperature S O C Medium Transfer the solution to a 15 ml snap cap tube i e Falcon and shake for at least 1 hour at 37 C to allow expression of the kanamycin resistance gene 5 Spread 20 100 ul from each transformation on a prewarmed selective plate and incubate overnight at 37 C To ensure even spreading of small volumes add 20 ul of S O C Medium We recommend that you plate two different volumes to ensure that at least one plate will have well spaced colonies 6 An efficient TOPO Cloning reaction may produce several hundred colonies Pick 5 10 colonies for analysis see Analyzing Transformants page 16 continued on next page 14 Transforming One Shot Competent E coli continued X 7 To prevent arcing of your samples during electroporation the volume of cells ei should be between 50 and 80 ul 0 1 cm cuvettes or 100 to 200 ul 0 2 cm cuvettes E E i If you experience arcing during transformation try one of the following suggestions e Reduce the voltage normally used to charge your electroporator by 10 e Reduce the pulse
6. The sequence of pENTR SD D TOPOS is available for downloading from our Web site www invitrogen com or by contacting Technical Service page 35 For more information about pENTR SD D TOPO see pages 30 31 M13 forward 20 priming site 501 TAACGCTAGC ATGGATGTTT TCCCAGTCAC GACGTTGTAA AACGACGGCC AGTCTTAAGC TCGGGCCCCA AATAATGATT 581 659 719 791 871 attL1 TTATTTTGAC TGATAGTGAC CTGTTCGTTG CAACAAATTG ATGAGCAATG CTTTTTTATA ATGCCAACT TTG gene 10 AAC Not translational enhancer RBS Leu r 1 f 1 AAA GCA GGC TCC GCG GCC GCC TTG TTT AAC TTT AAG AAG GAG CCC TTIG RACC NUCH AAG TTT CGT CCG AGG CGC CGG CGG AAC AAA TTG AAA TTC TTC CTC GGG AAG TG TIE Lys Ala Gly Ser Ala Ala Ala Leu Phe Asn Phe Lys Lys Glu Pro Phe Th Lys TAC ATG Tyr GGT SER Gly AAA TIT Lys GGG cee Gly Asc attL2 CGC GCC GAC CCA GET TTC TTG TAC AAAGTTGG CATTATAAGA AAGCATTGCT TATCAATTTG TTGCAACGAA GCG GG ClG CGT CGA AAG AAC VATG Arg Ala Asp Pro Ala Phe Leu Tyr T7 promoter priming site l r 1 CAGGTCACTA TCAGTCAAAA TAAAATCATT ATTTGCCATC CAGCTGATAT CCCCTATAGT GAGTCGTATT ACAT M13 reverse priming site AGCTGTTTCC TGGCAGCTCT m GGTCAT continued on next page Designing PCR Primers continued TOPO Cloning Site for pENTR TEV D TOPO Use the diagram below to help you design suitable PCR primers to clone your PCR product into pENTR TEV D TOPO Restriction sites are labeled to indicate
7. end Reverse PCR primer is complementary to the GTGG overhang at the 5 end Make sure that the reverse PCR primer does not contain the sequence CACC at the 5 end Large number of incorrect inserts cloned PCR cloning artifacts e Gel purify your PCR product to remove primer dimers and smaller PCR products e Optimize your PCR e Include a final extension step of 7 to 30 minutes during PCR Longer PCR products will need a longer extension time Incorrect PCR primer design Make sure that the forward and reverse PCR primers are designed correctly Few or no colonies obtained from sample reaction and the transformation control gave no colonies One Shot competent E coli stored incorrectly Store One Shot competent E coli at 80 C If you are using another E coli strain follow the manufacturer s instructions Did not perform the 1 hour grow out period before plating the transformation mixture After the heat shock step add S O C Medium and incubate the transformation mixture for 1 hour at 37 C before plating Insufficient amount of E coli plated Increase the amount of E coli plated Transformants plated on selective plates containing the wrong antibiotic Use the appropriate antibiotic for selection Appendix Performing the Control Reactions Introduction We recommend performing the following control TOPO Cloning reactions the first
8. proofreading polymerase and the appropriate PCR buffer Store Box 1 at 20 C Item Concentration Amount pENTR TOPO vector 15 20 ng ul linearized plasmid 20 ul TOPO adapted DNA in pENTR D TOPO or 50 glycerol pENTR SD D TOPO or 50 mM Tris HCI pH 7 4 at 25 C pENTR TEV D TOPO mM EDTA 2mMDTT 0 1 Triton X 100 100 ug ml BSA 30 uM bromophenol blue dNTP Mix 12 5 mM dATP 10 pl 12 5 mM dCTP 12 5 mM dGTP 12 5 mM dTTP in water pH 8 Salt Solution 1 2 M NaCl 50 ul 0 06 M MgCl Water ES 1ml M13 Forward 20 0 1 ug ul in TE Buffer pH 8 20 pl Sequencing Primer M13 Reverse Sequencing 0 1 ug ul in TE Buffer pH 8 20 pl Primer Control PCR Primers 0 1 ug ul each in TE Buffer pH 8 10 ul Control PCR Template 0 1 ug ul in TE Buffer pH 8 10 ul The table below provides the sequences of the M13 Forward 20 and M13 Reverse sequencing primers Primer Sequence pMoles Supplied M13 Forward 20 5 GTAAAACGACGGCCAG 3 407 M13 Reverse 5 CAGGAAACAGCTATGAC 3 385 continued on next page Kit Contents and Storage continued One Shot Reagents Genotype of E coli Strains Information for Non U S Customers Using Mach1 T1 Cells The following reagents are included with the One Shot TOP10 or Mach1 T18 Chemically Competent E coli kit Box 2 Transformation efficiency is 2 1 x 10 cfu ug plasmid DNA Store Box 2 at 80 C
9. Mach1 T1 chemically competent E coli supplied with the kit Box 2 e S O C Medium supplied with the kit Box 2 e pUC19 positive control to verify transformation efficiency if desired Box 2 e 42 C water bath or electroporator with cuvettes optional e 15 ml sterile snap cap plastic culture tubes for electroporation only e LB plates containing 50 ug ml kanamycin two for each transformation e LB plates containing 100 ug ml ampicillin if transforming pUC19 control e 37 C shaking and non shaking incubator There is no blue white screening for the presence of inserts Most transformants will contain recombinant plasmids with the PCR product of interest cloned in the correct orientation Sequencing primers are included in the kit to sequence across an insert in the multiple cloning site to confirm orientation and reading frame For each transformation you will need one vial of One Shot competent cells and two selective plates e Equilibrate a water bath to 42 C for chemical transformation or set up your electroporator if you are using electrocompetent E coli e Warm the vial of S O C Medium from Box 2 to room temperature e Warm selective plates at 37 C for 30 minutes e Thaw on ice one vial of One Shot cells from Box 2 for each transformation continued on next page 13 Transforming One Shot Competent E coli continued One Shot Use the following protocol to transform One Shot TOP10 o
10. interest in the entry construct with a Gateway destination vector Landy 1989 TEV recognition site Allows removal of the N terminal tag from your recombinant protein using AcTEV protease Carrington and Dougherty 1988 Dougherty et al 1988 TOPO Cloning site directional Allows rapid directional cloning of your PCR product 17 promoter priming site Allows in vitro transcription and sequencing of the insert M13 reverse priming site Allows sequencing of the insert Kanamycin resistance gene Allows selection of the plasmid in E coli pUC origin of replication ori Allows high copy replication and maintenance in E coli 33 Recipes LB Luria Bertani Medium and Plates 34 Composition 1 0 Tryptone 0 5 Yeast Extract 1 0 NaCl pH 7 0 1 For 1 liter dissolve 10 g tryptone 5 g yeast extract and 10 g NaCl in 950 ml deionized water 2 Adjust the pH of the solution to 7 0 with NaOH and bring the volume up to 1 liter 3 Autoclave on liquid cycle for 20 minutes Allow solution to cool to 55 C and add antibiotic if needed 4 Store at room temperature or at 4 C LB agar plates 1 Prepare LB medium as above but add 15 g L agar before autoclaving 2 Autoclave on liquid cycle for 20 minutes 3 After autoclaving cool to 55 C add antibiotic and pour into 10 cm plates 4 Let harden then invert and store at 4 C in the dark
11. into competent F coli and select for expression clones You may use any recA endA E coli strain including OmniMAX 2 T18 TOP10 DH5a or equivalent for transformation Do not transform the Gateway or MultiSite Gateway LR reaction mixture into E coli strains that contain the F episome e g TOP10F These strains contain the ccdA gene and will prevent negative selection with the ccdB gene continued on next page Guidelines to Perform the LR Recombination Reaction Performing the LR To perform the Gateway LR recombination reaction you will need Recombination Reaction Performing the MultiSite Gateway LR Recombination Reaction e Purified plasmid DNA of the entry clone containing your gene of interest e A destination vector of choice TM e LRClonase II enzyme mix see page x for ordering information TM e 2ug yul Proteinase K solution supplied with the LR Clonase II enzyme mix e TE Buffer pH 8 0 10 mM Tris HCI pH 8 0 1 mM EDTA e Appropriate chemically competent E coli host and growth media for expression e Appropriate selective plates For instructions to perform the LR recombination reaction refer to the Gateway Technology with Clonase II manual or to the manual for the destination vector you are using Before you can perform the MultiSite Gateway LR recombination reaction you will first need to generate 5 and 3 entry clones using Invitrogen s MultiSite Gateway Three F
12. pENTR TEVID The figure below shows the features of PENTR TEV D TOPO vector The TOPO Map complete sequence of pENTR TEV D TOPO is available for downloading from www invitrogen com or by contacting Technical Service see page 35 Comments for pENTR TEV D TOPO 2610 nucleotides rrnB T2 transcription termination sequence bases 268 295 rrnB T1 transcription termination sequence bases 427 470 M13 forward 20 priming site bases 537 552 attL1 bases 569 668 c TEV recognition site bases 689 709 TOPO recognition site bases 710 714 Overhang bases 715 718 TOPO recognition site bases 719 723 attL2 bases 735 834 T7 Promoter priming site bases 851 870 c M13 reverse priming site bases 875 891 Kanamycin resistance gene bases 1004 1813 pUC origin bases 1934 2607 c complementary sequence continued on next page 32 Map and Features of pENTR TEV D TOPO continued Features of pENTR TEV D TOPO 2610 bp contains the following elements Features pENTR TEVID have been functionally tested TOPO Feature Benefit rrnB T1 and T2 transcription termination sequences Reduces potential toxicity in E coli by preventing basal expression of the PCR product Orosz et al 1991 M13 forward 20 priming site Allows sequencing of the insert attL1 and attL2 sites Bacteriophage A derived recombination sequences that allow recombinational cloning of a gene of
13. problems obtaining transformants or the correct insert perform the Important control reactions described on page 23 25 or refer to the Troubleshooting section page 21 for tips to help you troubleshoot your experiment Long Term Once you have identified the correct clone be sure to purify the colony and make Storage a glycerol stock for long term storage We recommend that you store a stock of plasmid DNA at 20 C 1 Streak the original colony out for single colony on LB plates containing 50 ug ml kanamycin 2 Isolate a single colony and inoculate into 1 2 ml of LB containing 50 ug ml kanamycin 3 Grow until culture reaches stationary phase Mix 0 85 ml of culture with 0 15 ml of sterile glycerol and transfer to a cryovial 5 Store at 80 C 17 Guidelines to Perform the LR Recombination Reaction Introduction Important Destination Vectors E coli Host 18 Once you have obtained your entry clone you may e Perform an LR recombination reaction using Gateway LR Clonase II enzyme mix see page x for ordering information to transfer your gene of interest from the entry construct into any Gateway destination vector of choice to generate an expression clone e Perform a MultiSite Gateway LR recombination reaction with 5 and 3 entry clones the appropriate MultiSite Gateway destination vector and LR Clonase Plus enzyme mix see page x for ordering information to generate an expression clone
14. purification If you wish to purify your PCR product be extremely careful to remove all sources of nuclease contamination There are many protocols to isolate DNA fragments or remove oligonucleotides Refer to Current Protocols in Molecular Biology Unit 2 6 Ausubel et al 1994 for the most common protocols Three simple protocols are provided below The cloning efficiency may decrease with purification of the PCR product e g PCR product too dilute You may wish to optimize your PCR to produce a single band see Producing Blunt End PCR Products page 10 TM The S N A P Gel Purification Kit available from Invitrogen Catalog no K1999 25 allows you to rapidly purify PCR products from regular agarose gels 1 Electrophorese amplification reaction on a 1 to 5 regular TAE agarose gel Note Do not use TBE to prepare agarose gels Borate interferes with the sodium iodide step below 2 Cutout the gel slice containing the PCR product and melt it at 65 C in 2 volumes of the 6 M sodium iodide solution Add 1 5 volumes Binding Buffer Load solution no more than 1 ml at a time from Step 3 onto a S N A P column Centrifuge 1 minute at 3000 x g in a microcentrifuge and discard the supernatant TM If you have solution remaining from Step 3 repeat Step 4 Add 900 ul of the Final Wash Buffer Centrifuge 1 minute at full speed in a microcentrifuge and discard the flow through Repeat Step 7 Elute the purified PCR pr
15. recommend using the Control PCR Template and the Control PCR Primers included with the kit to perform the control reaction See the protocol on pages 23 25 for instructions Control Reaction vi Kit Contents and Storage Types of Kits This manual is supplied with the following kits Kit Size Catalog no pENTR D TOPO Cloning Kit with One Shot TOP10 Chemically Competent E coli 20reactions K2400 20 with One Shot Mach1 T1 Chemically Competent E coli 20reactions K2435 20 pENTR SD D TOPC Cloning Kit with One Shot TOP10 Chemically Competent E coli 20reactions K2420 20 with One Shot Mach1 T1 Chemically Competent E coli 20 reactions K2635 20 pENTR TEV D TOPO Cloning Kit with One Shot TOP10 Chemically Competent E coli 20reactions K2525 20 with One Shot Mach1 T1 Chemically Competent E coli 20reactions K2535 20 Shipping Storage Each pENTR Directional TOPO Cloning Kit is shipped on dry ice Each kit contains two boxes as described below Upon receipt store the boxes as detailed below Box Item Storage 1 pENTR TOPO Reagents 20 C 2 One Shot Chemically Competent E coli 80 C continued on next page vii Kit Contents and Storage continued pENTR TOPO Reagents Sequences of the Primers viii The following reagents are supplied with each pENTR TOPO vector Box 1 Note that the user must supply a thermostable
16. the actual cleavage site The shaded region corresponds to the DNA sequences that will be transferred from the entry clone into the destination vector following LR recombination The sequence of pENTR TEV D TOPO is available for downloading from our Web site www invitrogen com or by contacting Technical Service page 35 For more information about pENTR TEV D TOPO see pages 32 33 Note The sequence of the 5 TOPO recognition site has been changed from CCCTT to TCCTT resulting in an amino acid substitution of serine for proline This amino acid change increases the efficiency of TEV protease cleavage Kapust et al 2002 but does not affect the efficiency of TOPO Cloning M13 forward 20 priming site r 1 SS 501 TAACGCTAGC ATGGATGTTT TCCCAGTCAC GACGTTGTAA AACGACGGCC AGTCTTAAGC TCGGGCCCCA AATAATGATT attL1 581 TTATTTTGAC TGATAGTGAC CTGTTCGTTG CAACAAATTG ATGAGCAATG CTTTTTTATA ATGCCAACT TTG TAC AAA AAC ATG TTT Not TEV recognition site Leu Tyr Lys 659 720 AAA GCA GGC TCC TTT CGT COG AGG Lys Ala Gly Ser sel AAG GGT GGG CGC Tie Teck CCC Cee Lys Gly Gly Arg GCG CGC Ala GCC CGG Ala GCC CGG Ala GCC CGG Ala GTA CAT Val f CTC GAG GAA AAC CTG TAT TTT CAG GGC TCC TT eMPNeleMr wel GAG CTC CTT TTG GAC ATA AAA GTC CCG AGG AAG TGGRRXe Leu Glu Glu Asn Leu Tyr Phe ES dnd Ser Phe Thf attL2 TEV cleavage site r GAC CTG Asp CCA GGT Pro GCT CGA Ala TTC TTG TAC A
17. time you use the kit to help you evaluate your results Performing the control reactions involves producing a control PCR product using the reagents included in the kit and using this product directly in a TOPO Cloning reaction Before Starting For each transformation prepare two LB plates containing 50 ug ml kanamycin Producing the Use your thermostable proofreading polymerase and the appropriate buffer to Control PCR amplify the control PCR product Follow the manufacturer s recommendations Product for the proofreading polymerase you are using 1 To produce the 750 bp control PCR product set up the following 50 ul PCR Component Amount Control DNA Template 100 ng 1g 10X PCR Buffer appropriate for enzyme 5 ul dNTP Mix 0 5 ul Control PCR Primers 0 1 ug ul each 1u Sterile water 41 5 ul Proofreading polymerase 1 2 5 U l 1 ul Total volume 50 ul 2 Overlay with 70 ul 1 drop of mineral oil if required 3 Amplify using the following cycling parameters Step Time Temperature Cycles Initial Denaturation 2 minutes 94 C 1X Denaturation 1 minute 94 C Annealing 1 minute 55 C 25X Extension 1 minute 72 C Final Extension 7 minutes 72 C 1X 4 Remove 10 ul from the reaction and analyze by agarose gel electrophoresis Make sure that you see a single discrete 750 bp band 5 Estimate the concentration of the PCR product and adjust as necessary such that the a
18. to overnight For detailed instructions to set up the cleavage reaction refer to the manual included with the product TM e After cleavage remove AcTEV Protease from the reaction mixture using affinity chromatography on a nickel chelating resin e g ProBond Resin Catalog no K801 01 After digestion with TEV protease four vector encoded amino acids will remain at the N terminus of your recombinant protein Troubleshooting TOPO Cloning Reaction and Transformation pages 23 25 in parallel with your samples The table below lists some potential problems and possible solutions that may help you troubleshoot the TOPO Cloning and transformation reactions To help evaluate your results we recommend that you perform the control reactions see the transformation control gave colonies the TOPO Cloning reaction Problem Reason Solution Few or no colonies obtained Suboptimal ratio of PCR Use a 0 5 1 to 2 1 molar ratio of PCR from sample reaction and product TOPO vector used in product TOPO vector Too much PCR product used in the TOPO Cloning reaction e Dilute the PCR product e UseaO0 5 1 to 2 1 molar ratio of PCR product TOPO vector PCR primers contain 5 phosphates Do not add 5 phosphates to your PCR primers Incorrect PCR primer design e Make sure that the forward PCR primer contains the sequence CACC at the 5 end e Make sure that the reverse PCR pri
19. your entry clone with a Gateway destination vector When designing your forward PCR primer consider the following points below Refer to pages 8 9 for diagrams of the TOPO Cloning site for pENTR D TOPO pENTR SD D TOPO and pENTR TEV D TOPO To enable directional cloning the forward PCR primer must contain the sequence CACC at the 5 end of the primer The 4 nucleotides CACC base pair with the overhang sequence GTGG in each pENTR TOPO vector If you plan to express your PCR product in mammalian cells as a native or C terminal fusion tagged protein following recombination of the entry clone with a Gateway destination vector your sequence of interest should include a Kozak translation initiation sequence with an ATG initiation codon for proper initiation of translation Kozak 1987 Kozak 1991 Kozak 1990 An example of a Kozak consensus sequence is G AJ NNATGG Other sequences are possible but the G or A at position 3 and the G at position 4 are the most critical for function shown in bold The ATG initiation codon is underlined Note If your sequence of interest does not contain an initiation codon within the context of a Kozak sequence design the forward PCR primer to contain a Kozak sequence at the 5 end of the primer see Example on the next page If you plan to express your PCR product in mammalian cells as an N terminal fusion tagged protein following recombination of the entry clone with a Ga
20. your recombinant protein For more information about the Gateway Technology refer to the Gateway Technology with Clonase II manual which is available for downloading from www invitrogen com or by contacting Technical Service see page 35 continued on next page Overview continued MultiSite Gateway Technology Features of the pENTR TOPO Vectors The MultiSite Gateway Technology uses modifications of the site specific recombination reactions of the Gateway Technology see the previous page to allow simultaneous cloning of multiple DNA fragments in a defined order and orientation The MultiSite Gateway Three Fragment Vector Construction Kit available from Invitrogen Catalog no 12537 023 facilitates simultaneous cloning of DNA fragments in three entry vectors to create your own expression clone For more information about the MultiSite Gateway Technology and the MultiSite Gateway Three Fragment Vector Construction Kit refer to the MultiSite Gateway Three Fragment Vector Construction Kit manual which is available for downloading from our Web site or by contacting Technical Service The pENTR D TOPO pENTR SD D TOPO and pENTR TEV D TOPO vectors are designed to facilitate rapid directional TOPO Cloning of blunt end PCR products for entry into the Gateway System Features of the vectors include e attL1 and attL2 sites for site specific recombination of the entry clone with a Gateway destinati
21. 2 as would be the case if the 5 TOPO recognition site was encoded by CCCTT By changing the sequence of the 5 TOPO recognition site to TCCTT the first amino acid following the TEV recognition site is now serine This change does not affect TOPO Cloning efficiency and allows efficient TEV cleavage Experimental Outline Flow Chart The flow chart below describes the general steps required to produce and clone your blunt end PCR product Determine strategy for PCR Produce blunt end PCR product using properly designed PCR primers TOPO Cloning Reaction Mix together PCR product and pENTR TOPO vector Incubate 5 minutes at room temperature Transform into competent E coli cells Select and analyze colonies Choose a positive transformant and isolate plasmid DNA Proceed to the LR recombination reaction with a Gateway destination vector Methods Designing PCR Primers Designing Your PCR Primers Guidelines to Design the Forward PCR Primer The design of the PCR primers to amplify your gene of interest is critical for expression Depending on the pENTR TOPO vector you are using consider the following when designing your PCR primers Sequences required to facilitate directional cloning Sequences required for proper translation initiation of your PCR product Whether or not you wish your PCR product to be fused in frame with an N or C terminal tag after recombination of
22. 7 Kapust R B Tozser J Copeland T D and Waugh D S 2002 The P1 Specificity of Tobacco Etch Virus Protease Biochem Biophys Res Comm 294 949 955 Kozak M 1987 An Analysis of 5 Noncoding Sequences from 699 Vertebrate Messenger RNAs Nucleic Acids Res 15 8125 8148 Kozak M 1991 An Analysis of Vertebrate mRNA Sequences Intimations of Translational Control J Cell Biology 115 887 903 Kozak M 1990 Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic Ribosomes Proc Natl Acad Sci USA 87 8301 8305 Landy A 1989 Dynamic Structural and Regulatory Aspects of Lambda Site specific Recombination Ann Rev Biochem 58 913 949 Nayak S Li L and Lee J 2003 Enhanced TEV Protease Extends Enzyme Stability for Long Term Activity Focus 25 3 12 14 Olins P O Devine C S Rangwala S H and Kavka K S 1988 T7 Phage Gene 10 Leader RNA a Ribosome binding Site that Dramatically Enhances the Expression of Foreign Genes in Escherichia coli Gene 73 227 235 Orosz A Boros L and Venetianer P 1991 Analysis of the Complex Transcription Termination Region of the Escherichia coli rrnB Gene Eur J Biochem 201 653 659 Shuman S 1994 Novel Approach to Molecular Cloning and Polynucleotide Synthesis Using Vaccinia DNA Topoisomerase J Biol Chem 269 32678 32684 Shuman S 1991 Recombination Mediated by Vaccinia Virus DNA Topoi
23. AAGTTGGC ATTATAAGAA AGCATTGCTT ATCAATTTGT AAG AAC ATG Phe Leu Tyr T7 promoter priming site 1 791 TGCAACGAAC AGGTCACTAT CAGTCAAAAT AAAATCATTA TTTGCCATCC AGCTGATATC CCCTATAGTG AGTCGTATTA M13 reverse priming site r 1 871 CATGGTCATA GCTGTTTCCT GGCAGCTCTG Producing Blunt End PCR Products Introduction Materials Supplied by the User Producing Blunt End PCR Products Checking the PCR Product 10 Once you have decided on a PCR strategy and have synthesized the primers produce your blunt end PCR product using any thermostable proofreading polymerase Follow the guidelines below to produce your blunt end PCR product You will need the following reagents and equipment for PCR Note dNTPs adjusted to pH 8 are provided in the kit e Thermocycler and thermostable proofreading polymerase e 10X PCR buffer appropriate for your polymerase DNA template and primers to produce the PCR product Set up a 25 ul or 50 ul PCR reaction using the guidelines below e Follow the instructions and recommendations provided by the manufacturer of your thermostable proofreading polymerase to produce blunt end PCR products e Use the cycling parameters suitable for your primers and template Make sure to optimize PCR conditions to produce a single discrete PCR product e Usea7 to 30 minute final extension to ensure that all PCR products are completely extended e After cycling place the tube on ice or s
24. Invitrogen pENTR Directional TOPO Cloning Kits Five minute directional TOPO Cloning of blunt end PCR products into an entry vector for the Gateway System Catalog nos K2400 20 K2420 20 K2525 20 K2535 20 K2435 20 and K2635 20 Version G 6 April 2006 25 0434 A Limited Use Label License covers this product see Purchaser Notification By use of this product you accept the terms and conditions of the Limited Use Label License ii Table of Contents Table of Contents i sceau e HE OC Ro HR eb ettet ee e p ete t eee P e iii TOPO Cloning Procedure for Experienced DSerss escas ti tea piace eee edle based eund bn mund V Kit Contents and Storage edite Re oreet ite Pee Heine eee ib eae e Pope Eepe Pete Henn en festen eee eed vii Accessory Products isses EE Tanner emu Nam e eina aet x Introduction 1 Overview A 1 Low Directional TOPO Cloning Works sn nn i nel ie etel tabe aa iuda D dae 3 Experimental Outline st 55 om aine Adobe deu aatia umane nta dide 4 Lg n 5 Designing PER Primers sse osito eie ha eie tl etin bern data etie es 5 Producing Bl nt End PCR Products esee ete het ee RR ie er mn 10 Setting Up the TOPO Cloning Reaction usa re ae 11 Transforming One Shot Competent E coli unse 13 Analyzing Transformants yea een nenn pisei t ESA Eee tie o hri e IE Riese irte orante 16 Guidelines to Perform the LR Recombination Reaction eeeeeennenenenenssennnnnnnn 18 Guidelines to Perfor
25. OPO only or any N or C terminal tags if desired after recombination with the Gateway destination vector Amplify Your Gene of Interest Use a thermostable proofreading DNA polymerase and the PCR primers above to produce your blunt end PCR product Use agarose gel electrophoresis to check the integrity and determine the yield of your PCR product Perform the TOPO Cloning Reaction Set up the following TOPO Cloning reaction For optimal results use a 0 5 1 to 2 1 molar ratio of PCR product TOPO vector Reagent Chemical Electroporation Transformation Fresh PCR product 0 5 to4 ul 0 5 to 4 ul Salt solution Tul Dilute salt solution 1 4 Tul Water to a final volume of 5 ul to a final volume of 5 ul TOPO vector 1g Tul Total volume 6 ul 6 ul Mix gently and incubate for 5 minutes at room temperature Place on ice and proceed to transform One Shot chemically competent E coli below Transform One Shot Add 2 ul of the TOPO Cloning reaction into a vial of One Shot chemically Chemically Competent competent E coli cells and mix gently E coli Incubate on ice for 5 to 30 minutes Heat shock the cells for 30 seconds at 42 C without shaking Immediately transfer the tube to ice Add 250 ul of room temperature S O C Medium Incubate at 37 C for 1 hour with shaking Spread 50 200 ul of bacterial culture on a prewarmed selective plate and incubate overnight at 37 C We
26. amino acid such as glycine alanine or lysine Below is the sequence for the C terminus of a theoretical protein The stop codon is underlined GCG GTT AAG TCG GAG CAC TCG ACG ACT GCA TAG 3 e To fuse the ORF in frame with a C terminal tag supplied by the destination vector after recombination remove the stop codon by starting with nucleotides homologous to the last codon TGC and continue upstream The reverse primer will be 5 TGC AGT CGT CGA GTG CTC CGA CTT 3 This will amplify the C terminus without the stop codon and allow you to join the ORF in frame with a C terminal tag e If you don t want to join the ORF in frame with a C terminal tag simply design the reverse primer to include the stop codon 5 CTA TGC AGT CGT CGA GTG CTC CGA CTT 3 e Remember that the pENTR TOPO vectors accept blunt end PCR products e Donotadd 5 phosphates to your primers for PCR This will prevent ligation into the pENTR TOPO vectors e We recommend that you gel purify your oligonucleotides especially if they are long gt 30 nucleotides continued on next page Designing PCR Primers continued TOPO Cloning Site for pENTR D TOPO Use the diagram below to help you design suitable PCR primers to clone your PCR product into pENTR D TOPO Restriction sites are labeled to indicate the actual cleavage site The shaded region corresponds to the DNA sequences that will be transferred from the
27. ature or 4 C 12 1 Mix reaction gently and incubate for 5 minutes at room temperature 22 23 C Note For most applications 5 minutes will yield a sufficient number of colonies for analysis Depending on your needs the length of the TOPO Cloning reaction can be varied from 30 seconds to 30 minutes For routine subcloning of PCR products 30 seconds may be sufficient For large PCR products gt 1 kb or if you are TOPO Cloning a pool of PCR products increasing the reaction time may yield more colonies 2 Place the reaction on ice and proceed to Transforming One Shot Competent E coli next page Note You may store the TOPO Cloning reaction at 20 C overnight Transforming One Shot Competent E coli Introduction Materials Needed Note Preparing for Transformation Once you have performed the TOPO Cloning reaction you will transform your pENTR TOPO construct into competent E coli One Shot TOP10 or Mach1 T1 Chemically Competent E coli Box 2 are included with the kit to facilitate transformation however you may also transform electrocompetent cells see page x for ordering information Protocols to transform chemically competent or electrocompetent F coli are provided in this section In addition to general microbiological supplies i e plates spreaders you will need the following reagents and equipment e TOPO Cloning reaction from Step 2 previous page e One Shot TOP10 or
28. cells provided or electrocompetent cells see page x for ordering information e If you are transforming chemically competent E coli use the stock Salt Solution as supplied and set up the TOPO Cloning reaction as directed below e If you are transforming electrocompetent E coli the amount of salt in the TOPO Cloning reaction must be reduced to 50 mM NaCl 2 5 mM MgCl to prevent arcing during electroporation Dilute the stock Salt Solution 4 fold with water to prepare a 300 mM NaCl 15 mM MgCl Dilute Salt Solution Use the Dilute Salt Solution to set up the TOPO Cloning reaction as directed below Use the procedure below to perform the TOPO Cloning reaction Set up the TOPO Cloning reaction depending on whether you plan to transform chemically competent E coli or electrocompetent E coli Reminder For optimal results be sure to use a 0 5 1 to 2 1 molar ratio of PCR product TOPO vector in your TOPO Cloning reaction Note The blue color of the TOPO vector solution is normal and is used to visualize the solution Reagents Chemically Competent E coli Electrocompetent E coli Fresh PCR product 0 5 to 4 ul 0 5 to 4 ul Salt Solution 1 ul Dilute Salt Solution 1 4 1 ul Sterile Water add to a final volume of 5 ul add to a final volume of 5 ul TOPO vector 1 pl 1 ul Final volume 6 ul 6 ul Store all reagents at 20 C when finished Salt solution and water can be stored at room temper
29. e the transformations isolate plasmid DNA and digest with the appropriate restriction enzyme as listed below The table below lists the digestion patterns that you should see for inserts that are cloned in the correct orientation or in the reverse orientation Vector Restriction Expected Digestion Patterns bp Enzyme pENTR D TOPO Not I Correct orientation 127 3203 Reverse orientation 646 2684 Empty vector 2580 pENTR SD D Not I Correct orientation 148 3203 TOPO Reverse orientation 667 2684 Empty vector 2601 pENTR TEV D EcoR V Pst I Correct orientation 757 2602 TOPO Reverse orientation 250 3109 Empty vector 2610 Greater than 90 of the colonies should contain the 750 bp insert in the correct orientation Relatively few colonies should be produced in the vector only reaction pUC19 plasmid is included to check the transformation efficiency of the One Shot competent cells Transform one vial of One Shot competent cells with 10 pg of pUC19 using the protocol on page 14 Plate 10 ul of the transformation mixture plus 20 ul of S O C Medium on LB plates containing 100 ug ml ampicillin Transformation efficiency should be 2 1 x 10 cfu ug DNA 25 Gel Purifying PCR Products Introduction Note Using the S N A P Gel Purification Kit Quick S N A P Method 26 Smearing multiple banding primer dimer artifacts or large PCR products 73 kb may necessitate gel
30. ecause of mispriming or contaminating template Materials Needed e PCR Super Mix High Fidelity Invitrogen Catalog no 10790 020 e Appropriate forward and reverse PCR primers 20 uM each Procedure 1 Foreach sample aliquot 48 ul of PCR SuperMix High Fidelity into a 0 5 ml microcentrifuge tube Add 1 ul each of the forward and reverse PCR primer 2 Pick 5 10 colonies and resuspend them individually in 50 ul of the PCR SuperMix containing PCR primers remember to make a patch plate to preserve the colonies for further analysis Incubate reaction for 10 minutes at 94 C to lyse cells and inactivate nucleases Amplify for 20 to 30 cycles For the final extension incubate at 72 C for 10 minutes Store at 4 C Qv guo Visualize by agarose gel electrophoresis Once you have identified the correct clone s you may sequence your construct to confirm that your gene is cloned in the correct orientation Use the M13 Forward 20 and M13 Reverse included to help you sequence your insert see the diagrams on pages 8 9 for the location of the priming sites in each pENTR TOPO vector For the complete sequence of each pENTR TOPO vector see our Web site www invitrogen com or call Technical Service see page 35 Note The M13 Forward 20 and M13 Reverse primers are available separately from Invitrogen see page x for ordering information continued on next page Analyzing Transformants continued If you have
31. efficient expression of your gene of interest after recombination with a Gateway destination vector pENTR SD D TOPO Contains a T7 gene 10 translational enhancer and a ribosome binding site RBS for optimal expression of native protein after recombination with a prokaryotic Gateway destination vector Note Also suitable for efficient expression of your gene of interest in other hosts after recombination with a Gateway destination vector e g mammalian insect yeast pENTR TEV D Contains a Tobacco Etch Virus TEV recognition site TOPO for efficient TEV protease dependent cleavage of an N terminal tag from your recombinant protein after recombination and expression from a Gateway destination vector The Gateway Technology is a universal cloning method that takes advantage of the site specific recombination properties of bacteriophage lambda Landy 1989 to provide a rapid and highly efficient way to move your gene of interest into multiple vector systems To express your gene of interest using the Gateway Technology simply 1 TOPO Clone your blunt end PCR product into one of the pENTR TOPO vectors to generate an entry clone 2 Generate an expression construct by performing an LR recombination reaction between the entry clone and a Gateway destination vector of choice 3 Introduce your expression construct into the appropriate host e g bacterial mammalian yeast insect and express
32. ence continued on next page 30 Map and Features of pENTR SD D TOPO continued Features of pENTR SD D TOPO 2601 bp contains the following elements Features pENTR SD D have been functionally tested TOPO Feature Benefit rrnB T1 and T2 transcription termination sequences Reduces potential toxicity in E coli by preventing basal expression of the PCR product Orosz et al 1991 M13 forward 20 priming site Allows sequencing of the insert 17 gene 10 translational enhancer Sequence from bacteriophage T7 gene 10 that optimizes translation initiation Olins et al 1988 Ribosome binding site RBS Optimally spaced from the TOPO Cloning site for efficient translation of the PCR product attL1 and attL2 sites Bacteriophage A derived recombination sequences that allow recombinational cloning of a gene of interest in the entry construct with a Gateway destination vector Landy 1989 TOPO Cloning site directional Allows rapid directional cloning of your PCR product T7 promoter priming site Allows in vitro transcription and sequencing of the insert M13 reverse priming site Allows sequencing of the insert Kanamycin resistance gene Allows selection of the plasmid in E coli pUC origin of replication ori Allows high copy replication and maintenance in E coli 31 Map and Features of pENTR TEV D TOPO
33. entry clone into the destination vector following LR recombi nation The sequence of pENTR D TOPO is available for downloading from our Web site www invitrogen com or by contacting Technical Service page 35 For more information about pENTR D TOPO see pages 28 29 M13 forward 20 priming site r 1 MT 501 TAACGCTAGC ATGGATGTTT TCCCAGTCAC GACGTTGTAA AACGACGGCC AGTCTTAAGC TCGGGCCCCA AATAATGATT attL1 581 TTATTTTGAC TGATAGTGAC CTGTTCGTTG CAACAAATTG ATGAGCAATG CTTTTTTATA ATGCCAACT TTG TAC AAA AAC ATG TTT Not Asc Leu Tyr Lys 659 AAA GCA GGC TCC GCG GCC GCC CCC TTIeEmPEwePWwMMM AAG GGT GGG CGC GCC GAC CCA GCT TTC TTG TTT CGT CCG AGG CGC CGG CGG GGG AAG TGGRBXe TTE CCA CCC GCG CGG CTG GGT CGA AAG AAC Lys Ala Gly Ser Ala Ala Ala Pro Phe Ths Lys Gly Gly Arg Ala Asp Pro Ala Phe Leu attL2 719 TAC AAAGTTGGC ATTATAAGAA AGCATTGCTT ATCAATTTGT TGCAACGAAC AGGTCACTAT CAGTCAAAAT AAAATCATTA ATG Tyr T7 promoter priming site M13 reverse priming site r 1 r 1 801 TTTGCCATCC AGCTGATATC CCCTATAGTG AGTCGTATTA CATGGTCATA GCTGTTTCCT GGCAGCTCTG TOPO Cloning Use the diagram below to help you design suitable PCR primers to clone your PCR Site for product into pENTR SD D TOPO Restriction sites are labeled to indicate the pENTR SD D actual cleavage site The shaded region corresponds to the DNA sequences that TOPO will be transferred from the entry clone into the destination vector following LR recombination
34. g directional TOPO Cloning we have found that the molar ratio of PCR product TOPO vector used in the reaction is critical to its success To obtain the highest TOPO Cloning efficiency use a 0 5 1 to 2 1 molar ratio of PCR product TOPO vector see figure below Note that the TOPO Cloning efficiency decreases significantly if the ratio of PCR product TOPO vector is 0 1 1 or gt 5 1 see figure below These results are generally obtained if too little PCR product is used i e PCR product is too dilute or if too much PCR product is used in the TOPO Cloning reaction If you have quantitated the yield of your PCR product you may need to adjust the concentration of your PCR product before proceeding to TOPO Cloning Tip For pENTR TOPO vectors using 1 5 ng of a 1 kb PCR product or 5 10 ng of a 2 kb PCR product in a TOPO Cloning reaction generally results in a suitable number of colonies _ 100 28 gt 8 o lt 50 BS 5 8 0 0 1 1 10 PCR Product Vector Molar Ratio continued on next page 11 Setting Up the TOPO Cloning Reaction continued Using Salt Solution in the TOPO Cloning Reaction Performing the TOPO Cloning Reaction You will perform TOPO Cloning in a reaction buffer containing salt i e using the stock salt solution provided in the kit Note that the amount of salt added to the TOPO Cloning reaction varies depending on whether you plan to transform chemically competent
35. itrogen com Purchaser Notification continued Limited Use Label License No 19 Gateway Cloning Products Gateway Clone Distribution Policy This product and its use is the subject of one or more of U S Patent Nos 5 888 732 6 143 557 6 171 861 6 270 969 and 6 277 608 and or other pending U S and foreign patent applications owned by Invitrogen Corporation The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The purchase of this product does not convey a license under any method claims in the foregoing patents or patent applications or to use this product with any recombination sites other than those purchased from Invitrogen Corporation or its authorized distributor The right to use methods claimed in the foregoing patents or patent applications with this product for research purposes only can only be acquired by the use of Clonase purchased from Invitrogen Corporation or its authorized distributors The buyer cannot modify the recombination sequence s contained in this product for any purpose The buyer cannot sell or otherwise transfer a this product b its components or c materials made by the employment of this product or its components to a third party or otherwise use this product or its components or materials made by the emplo
36. l technology in their research Use of Invitrogen s Gateway Technology including Gateway clones for purposes other than scientific research may require a license and questions concerning such commercial use should be directed to Invitrogen s licensing department at 760 603 7200 Product Qualification Introduction Vectors TOPO Cloning Efficiency Primers One Shot Chemically Competent E coli This section describes the criteria used to qualify the components of the pENTR TOPO Cloning Kits Prior to adaptation with topoisomerase I each supercoiled pENTR vector is qualified by e Performing restriction enzyme digestion to verify its structure e Performing an LR recombination reaction with a Gateway destination vector to confirm its functionality After adaptation with topoisomerase I each pENTR TOPO vector is lot qualified using the control reagents included in the kit Under conditions described on pages 23 25 a 750 bp control PCR product is amplified using a forward primer containing CACC at its 5 end and a reverse primer The PCR product is TOPO Cloned into the pENTR TOPO vector and transformed into the One Shot TOP10 chemically competent E coli included with the kit Each lot of vector should yield greater than 90 cloning efficiency At least 10 transformants are characterized using restriction digest Of the transformants characterized greater than 90 should contain an inse
37. length by reducing the load resistance to 100 ohms e Ethanol precipitate the TOPO Cloning reaction and resuspend in water prior to electroporation 15 Analyzing Transformants Analyzing Positive 1 Pick 5 10 colonies and culture them overnight in LB or SOB medium Clones Analyzing Transformants by PCR Sequencing 16 containing 50 100 ug ml kanamycin Note If you transformed One Shot Mach1 T1 competent E coli you may inoculate overnight grown colonies and culture them for only 4 hours in pre warmed LB medium containing 50 ug ml kanamycin before isolating plasmid DNA For optimal results inoculate as much of a single colony as possible 2 Isolate plasmid DNA using your method of choice If you need ultra pure plasmid DNA for automated or manual sequencing we recommend using Invitrogen s PureLink HQ Mini Plasmid Purification Kit Catalog no K2100 01 3 Analyze the plasmids by restriction analysis or PCR see below to confirm the presence and correct orientation of the insert Use the protocol below or any other suitable protocol to analyze positive transformants using PCR For PCR primers use a combination of the M13 Forward 20 primer or the M13 Reverse primer and a primer that hybridizes within your insert You will have to determine the amplification conditions If you are using this technique for the first time we recommend performing restriction analysis in parallel Artifacts may be obtained b
38. m TEV Cleavage of Recombinant Proteins 20 Troubli shoolting eoe tete ER Ce anced DLE 21 Peli qme M 23 Performing the Control Reactions ssssssssssssseseeeeeenenenenette een aria EE E tenente tenerent 23 Gel Purifying PCR Products tennenenenneneneteertti tete tenete tenete nter tenete tentent 26 Map and Featuresof DENTR DETOPQS 2 28 Map and Features of pENTR SD D TOPO a euer 30 Map and Peatures of PENTR TEV 7D TOPO essen 32 RECIPES sess scree P 34 Technical Service mn eMe 35 P rch set Notification e cete eet een e n he e eee ese ri tatoo cies 36 Gateway Clone Distribution Policy seeren m oreen tin Reh 38 Product Qualification Rete eie eset ti t Mi re P RR OU aeaa aA bete ER 39 Retereticessciceote einen atender nni Ar nie En reto eee la 40 iii iv TOPO Cloning Procedure for Experienced Users This quick reference sheet is provided for experienced users of the TOPO Cloning procedure If you are performing the TOPO Cloning procedure for the first time we recommend that you follow the detailed protocols provided in the manual Introduction Step Action Design PCR Primers Include the 4 base pair sequences CACC necessary for directional cloning on the 5 end of the forward primer Design the primers such that your gene of interest will be optimally expressed and fused in frame with the TEV recognition site in pENTR TEV D T
39. mer does not contain the sequence CACC at the 5 end Used Taq polymerase or a Tag proofreading polymerase mixture for PCR Use a proofreading polymerase for PCR Large PCR product e Use a 0 5 1 to 2 1 molar ratio of PCR product TOPO vector Increase the incubation time of the TOPO reaction from 5 minutes to 30 minutes e Gel purify the PCR product to remove primer dimers and other artifacts PCR reaction contains artifacts i e does not run as a single discrete band on an agarose gel Optimize your PCR using the proofreading polymerase of your choice e Gel purify your PCR product continued on next page 21 Troubleshooting continued TOPO Cloning Reaction and Transformation continued 22 Problem Reason Solution Few or no colonies obtained from sample reaction and the transformation control gave colonies continued Cloning large pool of PCR products or a toxic gene e Increase the incubation time of the TOPO reaction from 5 minutes to 30 minutes e Usea0 5 1to 2 1 molar ratio of PCR product TOPO vector Incomplete extension during PCR Include a final extension step of 7 to 30 minutes during PCR Longer PCR products will need a longer extension time Large percentage of inserts cloned in the incorrect orientation Incorrect PCR primer design Make sure that the forward PCR primer contains the sequence CACC at the 5
40. mount of PCR product used in the control TOPO Cloning reaction results in an optimal molar ratio of PCR product TOPO vector i e 0 5 1 to 2 1 Proceed to the Control TOPO Cloning Reactions next page continued on next page 23 Performing the Control Reactions continued Control TOPO Cloning Reactions 24 Using the control PCR product produced on the previous page and the pENTR TM TOPO vector set up two 6 ul TOPO Cloning reactions as described below If you plan to transform electrocompetent E coli use Dilute Salt Solution in place of the Salt Solution 1 Setup control TOPO Cloning reactions Reagent Vector Only Vector PCR Insert Water 4 ul 3 pl Salt Solution 1u Tul Control PCR Product 1 ul pENTR D TOPO vector 1 ul 1 pl Total volume 6 ul 6 ul 2 Incubate at room temperature for 5 minutes and place on ice 3 Transform 2 ul of each reaction into separate vials of One Shot competent cells using the protocol on page 14 4 Spread 50 200 ul of each transformation mix onto LB plates containing 50 ug ml kanamycin Be sure to plate two different volumes to ensure that at least one plate has well spaced colonies 5 Incubate overnight at 37 C continued on next page Performing the Control Reactions continued Analysis of Results Transformation Control Hundreds of colonies from the vector PCR insert reaction should be produced To analyz
41. nant protein Guidelines to perform TEV cleavage are provided in this section For highly efficient TEV protease directed cleavage we recommend using AcTEV Protease available from Invitrogen Catalog nos 12575 015 and 12575 023 AcTEV Protease is an enhanced form of TEV protease that is highly site specific active and more stable than native TEV protease Nayak et al 2003 Following digestion ACTEV Protease may be easily removed from the cleavage reaction by affinity chromatography using the polyhistidine 6xHis tag at the N terminus of the protease TM General guidelines to use ACTEV Protease for cleavage are provided below For detailed instructions and recommendations to optimize cleavage refer to the manual included with the product TM One unit of ACTEV Protease cleaves gt 85 of 3 ug of a control substrate in 1 hour at 30 C Follow the guidelines below when using AcTEV Protease e For optimal yield of cleaved recombinant protein partially purify or purify recombinant fusion protein before performing cleavage e Use the following digestion conditions as a starting point and optimize the cleavage reaction as necessary by varying the amount of AcTEV Protease incubation temperature or reaction time TM For a cleavage reaction using 20 ug of fusion protein use 10 units of ACTEV Protease in a reaction volume of 150 ul Incubate the reaction mixture at 30 C for 1 hour or at 4 C for 4 hours
42. nufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that none of i this product ii any of its components or iii a method claim of the foregoing patents was used in the manufacture of such product Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the use of this product to manufacture a protein for sale provided that no method claim in the above patents was used in the manufacture of such protein If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to use this product for purposes other than those permitted above contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 For additional information about Invitrogen s policy for the use and distribution of Gateway clones see the section entitled Gateway Clone Distribution Policy next page 37 Gateway Clone Distribution Policy Introduction Gateway Entry Clones Gateway Expression Clones Additional Terms and Conditions 38 The information supplied in this section is intended to provide clarity concerning Invitrogen s policy for the use and distributi
43. oduct in 40 ul of TE or sterile water Use 4 ul for the TOPO Cloning reaction and proceed as described on page 12 An even easier method is to simply cut out the gel slice containing your PCR product place it on top of the S N A P column bed and centrifuge at full speed for 10 seconds Use 1 2 ul of the flow through in the TOPO Cloning reaction page 12 Be sure to make the gel slice as small as possible for best results continued on next page Gel Purifying PCR Products continued Low Melt Agarose Method Note If you prefer to use low melt agarose use the procedure below Note that gel purification will result in a dilution of your PCR product and a potential loss of cloning efficiency 1 Electrophorese as much as possible of your PCR reaction on a low melt agarose gel 0 8 to 1 2 in TAE buffer Visualize the band of interest and excise the band Place the gel slice in a microcentrifuge tube and incubate the tube at 65 C until the gel slice melts Place the tube at 37 C to keep the agarose melted Add 4 ul of the melted agarose containing your PCR product to the TOPO Cloning reaction as described on page 12 Incubate the TOPO Cloning reaction at 37 C for 5 to 10 minutes This is to keep the agarose melted Transform 2 to 4 ul directly into One Shot competent cells using the method on page 14 The cloning efficiency may decrease with purification of the PCR product You may wish to optimi
44. of interest in the entry construct with a Gateway destination vector Landy 1989 TOPO Cloning site directional Allows rapid directional cloning of your PCR product 17 promoter priming site Allows in vitro transcription and sequencing of the insert M13 reverse priming site Allows sequencing of the insert Kanamycin resistance gene Allows selection of the plasmid in E coli pUC origin of replication ori Allows high copy replication and maintenance in E coli 29 Map and Features of pENTR SD D TOPO pENTR SD D The figure below shows the features of pENTR SD D TOPO vector The TOPO Map complete sequence of pENTR SD D TOPO is available for downloading from www invitrogen com or by contacting Technical Service see page 35 Comments for pENTR SD D TOPO 2601 nucleotides rrnB T2 transcription termination sequence bases 268 295 rrnB T1 transcription termination sequence bases 427 470 M13 forward 20 priming site bases 537 552 attL1 bases 569 668 c T7 gene 10 translational enhancer bases 684 692 Ribosome binding site bases 694 700 TOPO recognition site 1 bases 701 705 Overhang bases 706 709 TOPO recognition site 2 bases 710 714 attL2 bases 726 825 T7 Promoter priming site bases 842 861 c M13 reverse priming site bases 866 882 Kanamycin resistance gene bases 995 1804 pUC origin bases 1925 2598 c complementary sequ
45. on of cloned nucleic acid fragments including open reading frames created using Invitrogen s commercially available Gateway Technology Invitrogen understands that Gateway entry clones containing atfL1 and attL2 sites may be generated by academic and government researchers for the purpose of scientific research Invitrogen agrees that such clones may be distributed for scientific research by non profit organizations and by for profit organizations without royalty payment to Invitrogen Invitrogen also understands that Gateway expression clones containing attB1 and attB2 sites may be generated by academic and government researchers for the purpose of scientific research Invitrogen agrees that such clones may be distributed for scientific research by academic and government organizations without royalty payment to Invitrogen Organizations other than academia and government may also distribute such Gateway expression clones for a nominal fee 10 per clone payable to Invitrogen We would ask that such distributors of Gateway entry and expression clones indicate that such clones may be used only for research purposes that such clones incorporate the Gateway Technology and that the purchase of Gateway Clonase from Invitrogen is required for carrying out the Gateway recombinational cloning reaction This should allow researchers to readily identify Gateway containing clones and facilitate their use of this powerfu
46. on vector e T7 gene 10 translation enhancer and ribosome binding site for efficient translation of the PCR product in prokaryotes pENTR SD D TOPO only e TEV recognition site for TEV protease dependent cleavage of an N terminal tag from your recombinant protein pENTR TEV D TOPO only e Directional TOPO Cloning site for rapid and efficient directional cloning of blunt end PCR products see page 3 for more information e r nB transcription termination sequences to prevent basal expression of the PCR product of interest in E coli e Kanamycin resistance gene for selection in E coli e pUC origin for high copy replication and maintenance of the plasmid in E coli How Directional TOPO Cloning Works How Topoisomerase I Works Directional TOPO Cloning Note Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites CCCTT see Note below and cleaves the phosphodiester backbone in one strand Shuman 1991 The energy from the broken phosphodiester backbone is conserved by formation of a covalent bond between the 3 phosphate of the cleaved strand and a tyrosyl residue Tyr 274 of topoisomerase I The phospho tyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5 hydroxyl of the original cleaved strand reversing the reaction and releasing topoisomerase Shuman 1994 TOPO Cloning exploits this reaction to efficiently clone PCR products Directional joining of double st
47. r Mach1 T1 Chemical chemically competent E coli Transformation 1 Add 2 ul of the TOPO Cloning reaction from Performing the TOPO Protocol Cloning Reaction Step 2 page 12 into a vial of One Shot Chemicall 8 P pag y Competent E coli and mix gently Do not mix by pipetting up and down Note If you are transforming the pUC19 control plasmid use 10 pg 1 ul 2 Incubate on ice for 5 to 30 minutes Note Longer incubations on ice seem to have a minimal effect on transformation efficiency The length of the incubation is at the user s discretion Heat shock the cells for 30 seconds at 42 C without shaking Immediately transfer the tubes to ice Add 250 ul of room temperature S O C Medium Cap the tube tightly and shake the tube horizontally 200 rpm at 37 C for 1 hour DT PY 7 Spread 50 200 ul from each transformation on a prewarmed selective plate and incubate overnight at 37 C We recommend that you plate two different volumes to ensure that at least one plate will have well spaced colonies 8 An efficient TOPO Cloning reaction may produce several hundred colonies Pick 5 10 colonies for analysis see Analyzing Transformants page 16 Transformation by Use ONLY electrocompetent cells for electroporation to avoid arcing Do not Electroporation use the One Shot TOP10 or Mach1 T1 chemically competent cells for electroporation 1 Add2 ul of the TOPO Cloning reaction from Performing the TOPO Cloning Reaction
48. r implied including any warranty of merchantability or fitness for a particular purpose 35 Purchaser Notification Introduction Information for European Customers Limited Use Label License No 5 Invitrogen Technology 36 Use of the pENTR Directional TOPO Cloning Kits is covered under the licenses detailed below The Mach1 T1 E coli strain is genetically modified to carry the lacZAM15 hsdR lacX74 recA endA tonA genotype As a condition of sale use of this product must be in accordance with all applicable local legislation and guidelines including EC Directive 90 219 EEC on the contained use of genetically modified organisms The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third pa
49. ragment Vector Construction Kit Catalog no 12537 023 Once you have generated the 5 and 3 entry clones you will use the 5 and 3 entry clones the entry clone containing your gene of interest and the other reagents supplied in the MultiSite Gateway Three Fragment Vector Construction Kit including LR Clonase Plus enzyme mix and the pDEST R4 R3 destination vector in a MultiSite Gateway LR recombination reaction to generate an expression clone For instructions to generate 5 and 3 entry clones and to perform the MultiSite Gateway LR recombination reaction refer to the MultiSite Gateway Three Fragment Vector Construction Kit manual 19 Guidelines to Perform TEV Cleavage of Recombinant Proteins Introduction Obtaining TEV Protease AcTEV Protease Unit Definition General Guidelines to Use AcTEV Protease Note 20 If you have cloned your PCR product into pENTR TEV D TOPO your gene of interest will be fused in frame with a TEV recognition sequence Performing an LR recombination or MultiSite Gateway LR recombination using the pENTR TEV D TOPO entry clone and a suitable destination vector allows you to generate an expression clone containing a TEV cleavage site just upstream of the translation initiation site of your recombinant protein Once expressed you may generate nearly native protein by using TEV protease to cleave any N termi nal fusion tags or sequences e g the attL site from the recombi
50. rand DNA using TOPO charged oligonucleotides occurs by adding a 3 single stranded end overhang to the incoming DNA Cheng and Shuman 2000 This single stranded overhang is identical to the 5 end of the TOPO charged DNA fragment At Invitrogen this idea has been modified by adding a 4 nucleotide overhang sequence to the TOPO charged DNA and adapting it to a whole vector format In this system PCR products are directionally cloned by adding four bases to the forward primer CACC The overhang in the cloning vector GTGG invades the 5 end of the PCR product anneals to the added bases and stabilizes the PCR product in the correct orientation Inserts can be cloned in the correct orientation with efficiencies equal to or greater than 90 Topoisomerase 9 CCCTT CACC ATG NNN NNN AAG GG GGGAAGTGG GTGG TAC NNN NNN TTC CC PCR product Overhang 9 Overhang invades double stranded o DNA displacing the bottom strand Topoisomerase CCCTTCACC ATG NNN NNN AAG GG SGGERAGTGE TAC NNN NNN TTC CC S amp The 5 TOPO recognition site in pENTR TEV D TOPO is encoded by the sequence TCCTT rather than CCCTT This is because the 5 TOPO recognition site directly follows the TEV recognition site and studies have shown that TEV protease does not cleave efficiently if the first amino acid following the TEV recognition sequence is proline Kapust et al 200
51. rt in the correct orientation Primers are lot qualified by DNA sequencing experiments using the dideoxy chain termination technique One Shot TOP10 and Mach1 T1 chemically competent cells are tested for transformation efficiency using the control plasmid included in the kit Transformed cultures are plated on LB plates containing 100 ug ml ampicillin and the transformation efficiency is calculated Test transformations are performed in duplicate Transformation efficiency should be greater than 1x 10 cfu ug plasmid DNA In addition untransformed cells are tested for the appropriate antibiotic sensitivity and lack of phage contamination 39 References Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K 1994 Current Protocols in Molecular Biology New York Greene Publishing Associates and Wiley Interscience Carrington J C and Dougherty W G 1988 A Viral Cleavage Site Cassette Identification of Amino Acid Sequences Required for Tobacco Etch Virus Polyprotein Processing Proc Natl Acad Sci USA 85 3391 3395 Cheng C and Shuman S 2000 Recombinogenic Flap Ligation Pathway for Intrinsic Repair of Topoisomerase IB Induced Double Strand Breaks Mol Cell Biol 20 8059 8068 Dougherty W G Carrington J C Cary S M and Parks T D 1988 Biochemical and Mutational Analysis of a Plant Virus Polyprotein Cleavage Site EMBO J 7 1281 128
52. rty and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Invitrogen Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing inv
53. ry clone with a Gateway destination vector then include the native sequence containing the stop codon in the reverse primer or make sure the stop codon is upstream from the reverse PCR primer binding site see Example 2 on the next page continued on next page Designing PCR Primers continued Example 1 of Reverse Primer Design Example 2 of Reverse Primer Design Important Below is the sequence of the C terminus of a theoretical protein You want to fuse the protein in frame with a C terminal tag following recombination of the entry clone with a Gateway destination vector The stop codon is underlined DNA sequence AAG TCG GAG CAC TCG ACG ACG GTG TAG 3 One solution is to design the reverse PCR primer to start with the codon just up stream of the stop codon but the last two codons contain GTGG underlined below which is identical to the 4 bp overhang sequence As a result the reverse primer will be complementary to the 4 bp overhang sequence increasing the probability that the PCR product will clone in the opposite orientation You want to avoid this situation DNA sequence AAG TCG GAG CAC TCG ACG ACG GTG TAG 3 Proposed Reverse PCR primer sequence TG AGC TGC TGC CAC AAA 5 Another solution is to design the reverse primer so that it hybridizes just down stream of the stop codon but still includes the C terminus of the ORF Note that you will need to replace the stop codon with a codon for an innocuous
54. sequence see boxed sequence allowing proper translation initiation of the PCR product in mammalian cells e The ATG initiation codon is properly spaced from the RBS in pENTR SD D TOPO only allowing proper translation of the PCR product in prokaryotic cells The first three base pairs of the PCR product following the 5 CACC overhang will constitute a functional codon When designing your reverse PCR primer consider the following points below Refer to pages 8 9 for diagrams of the TOPO Cloning site for pENTR D TOPO pENTR SD D TOPOC and pENTR TEV D TOPO e To ensure that your PCR product clones directionally with high efficiency the reverse PCR primer MUST NOT be complementary to the overhang sequence GTGG at the 5 end A one base pair mismatch can reduce the directional cloning efficiency from 90 to 50 increasing the likelihood of your ORF cloning in the opposite orientation see Example 1 on the next page We have not observed evidence of PCR products cloning in the opposite orientation from a two base pair mismatch e If you wish to fuse your PCR product in frame with a C terminal tag following recombination of the entry clone with a Gateway destination vector then design the reverse PCR primer to remove the native stop codon in the gene of interest see Example 2 on the next page e If you do not wish to fuse your PCR product in frame with a C terminal tag following recombination of the ent
55. somerase I in Escherichia coli is Sequence Specific Proc Natl Acad Sci USA 88 10104 10108 02001 2006 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 40 41 invitrogen Corporate Headquarters Invitrogen Corporation 1600 Faraday Avenue Carlsbad CA 92008 T 1 760 603 7200 F 1760 602 6500 E tech service invitrogen com For country specific contact information visit our web site at www invitrogen com
56. teway destination vector your sequence of interest does not need to contain a Kozak translation initiation sequence A Kozak sequence is provided by the appropriate destination vector Note In this case internal initiation may occur if your PCR product contains an endogenous Kozak sequence If you plan to express your PCR product in prokaryotic cells without an N terminal fusion tag following recombination of the entry clone with a Gateway destination vector you should TOPO Clone your PCR product into pENTR SD D TOPO pENTR SD D TOPO contains a 17 gene 10 translational enhancer and a ribosome binding site RBS to enable efficient translation of the PCR product in E coli To ensure optimal spacing for proper translation design your forward PCR primer so that the ATG initiation codon of your PCR product directly follows the CACC necessary for directional cloning see Example on the next page continued on next page Designing PCR Primers continued Example of Forward Primer Design Note Guidelines to Design the Reverse Primer Below is the DNA sequence of the N terminus of a theoretical protein and the proposed sequence for your forward PCR primer The ATG initiation codon is underlined DNA sequence 5 ATG GGA TCT GAT AAA Proposed Forward PCR primer 5 C ACC ATG GGA TCT GAT AAA If you design the forward PCR primer as noted above then e The ATG initiation codon falls within the context of a Kozak
57. to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Service Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Service Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed o
58. tore at 20 C for up to 2 weeks Proceed to Checking the PCR Product below After you have produced your blunt end PCR product use agarose gel electrophoresis to verify the quality and quantity of your PCR product Check for the following outcomes below e Be sure you have a single discrete band of the correct size If you do not have a single discrete band follow the manufacturer s recommendations to optimize your PCR with the polymerase of your choice Alternatively you may gel purify the desired product see pages 26 27 e Estimate the concentration of your PCR product You will use this information when setting up your TOPO Cloning reaction see Amount of PCR Product to Use in the TOPO Cloning Reaction next page for details Setting Up the TOPO Cloning Reaction Introduction Amount of PCR Product to Use in the TOPO Cloning Reaction Once you have produced the desired blunt end PCR product you are ready to TOPO Clone it into the pENTR TOPO vector and transform the recombinant vector into One Shot competent E coli You should have everything you need set up and ready to use to ensure that you obtain the best possible results We suggest that you read this section and the section entitled Transforming One Shot Competent E coli pages 13 14 before beginning If this is the first time you have TOPO Cloned perform the control reactions on pages 23 25 in parallel with your samples When performin
59. yment of this product or its components for Commercial Purposes The buyer may transfer information or materials made through the employment of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Notwithstanding the preceding any buyer who is employed in an academic or government institution may transfer materials made with this product to a third party who has a license from Invitrogen under the patents identified above to distribute such materials Transfer of such materials and or information to collaborators does not convey rights to practice any methods claimed in the foregoing patents or patent applications Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the ma
60. ze your PCR to produce a single band 27 Map and Features of pENTR D TOPO pENTR D TOPO The figure below shows the features of pENTR D TOPO vector The complete Map sequence of pENTR D TOPO is available for downloading from www invitrogen com or by contacting Technical Service see page 35 Comments for pENTR D TOPO 2580 nucleotides rrnB T2 transcription termination sequence bases 268 295 rrnB T1 transcription termination sequence bases 427 470 M13 forward 20 priming site bases 537 552 attL1 bases 569 668 c TOPO recognition site 1 bases 680 684 Overhang bases 685 688 TOPO recognition site 2 bases 689 693 attL2 bases 705 804 T7 Promoter priming site bases 821 840 c M13 reverse priming site bases 845 861 Kanamycin resistance gene bases 974 1783 pUC origin bases 1904 2577 c complementary sequence continued on next page 28 Map and Features of pENTR D TOPO continued Features of pENTR D TOPO 2580 bp contains the following elements Features have pENTR D TOPO been functionally tested Feature Benefit rrnB T1 and T2 transcription termination sequences Reduces potential toxicity in E coli by preventing basal expression of the PCR product M13 forward 20 priming site Allows sequencing of the insert attL1 and attL2 sites Bacteriophage A derived recombination sequences that allow recombinational cloning of a gene

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