Invitrogen Topo TA kit
Contents
1. For each transformation you will need one vial of competent cells and two selective plates e Equilibrate a water bath to 42 C 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 see Important note below e Spread 40 ul of 40 mg ml X gal on each LB plate and incubate at 37 C until ready for use e Thaw on ice 1 vial of One Shot cells for each transformation If you are performing the rapid chemical transformation protocol or if you wish to visualize colonies within 8 hours of plating it is essential that you prewarm your LB plates containing 50 100 pg ml ampicillin prior to spreading continued on next page Transforming One Shot Mach1 T1 Competent Cells continued TM One Shot For optimal growth of Mach1 T1 E coli cells it is essential that selective plates Chemical are prewarmed to 37 C prior to spreading Transformation 1 Protocol o gg Add 2 ul of the TOPO Cloning reaction from Performing the TOPO Cloning Reaction Step 2 page 5 into a vial of One Shot Chemically Competent E coli and mix gently Do not mix by pipetting up and down Incubate on ice for 5 to 30 minutes Note Longer incubations on ice do not seem to have any affect 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 ice2. 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 serviceGinvitrogen com E mail jpinfo invitrogen com E mail MSDS Limited Warranty 24 eurotech invitrogen com MSDSs Material Safety Data Sheets are available on our website at www invitrogen com msds Invitrogen is committed to providing our customers with high quality goods and services Our goal is 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
3. Cut the area of the gel containing the desired DNA fragment using a clean sharp blade Minimize the amount of surrounding agarose excised with the fragment Weigh the gel slice Add Gel Solubilization Buffer GS1 supplied in the kit as follows e For lt 2 agarose gels place up to 400 mg gel into a sterile 1 5 ml polypropylene tube Divide gel slices exceeding 400 mg among additional tubes Add 30 ul Gel Solubilization Buffer GS1 for every 10 mg of gel e For gt 2 agarose gels use sterile 5 ml polypropylene tubes and add 60 ul Gel Solubilization Buffer GS1 for every 10 mg of gel Incubate the tube at 50 C for 15 minutes Mix every 3 minutes to ensure gel dissolution After gel slice appears dissolved incubate for an additional 5 minutes Preheat an aliquot of TE Buffer TE to 65 70 C Place a Quick Gel Extraction Column into a Wash Tube Pipette the mixture from Step 5 above onto the column Use 1 column per 400 mg agarose Centrifuge at gt 12 000 x g for 1 minute Discard the flow through Place the column back into the Wash Tube Optional Add 500 ul Gel Solubilization Buffer GS1 to the column Incubate at room temperature for 1 minute Centrifuge at gt 12 000 x g for 1 minute Discard the flow through Place the column back into the Wash Tube Add 700 ul Wash Buffer W9 with ethanol add 96 100 ethanol to the Wash Buffer according to instructions on the label of the bottle to the column and incubate at room t
4. LacZa fragment bases 1 547 M13 reverse priming site bases 205 221 Multiple cloning site bases 234 357 T7 promoter priming site bases 364 383 M13 Forward 20 priming site bases 391 406 f1 origin bases 548 985 Kanamycin resistance ORF bases 1319 2113 Ampicillin resistance ORF bases 2131 2991 pUC origin bases 3136 3809 kb 15 Map of pCR II TOPO pCR ll TOPO Map The map below shows the features of pCR II TOPO and the sequence surrounding the TOPO Cloning site Restriction sites are labeled to indicate the actual cleavage site The arrows indicate the start of transcription for Sp6 and T7 polymerases The complete sequence of pCR II TOPO is available for downloading from our Web site www invitrogen com or by contacting Technical Service page 24 16 lacZa ATG M13 Reverse Primer CAG GTC GAA CIT ACA GCT ATG ACC ATG TGT CGA TAC TGG TAC TAC ATG GCC CGG CCA GGT TCA AGT AGT TCA TCA AGT Nsil Hind IIl AGC TAT GCA TCA AGC TCG ATA CGT AGT TCG BstX EcoRI GTG CTG GAA TTC GCC CAC GAC CTT AAG CGG BstX CAC TGG CGG CCG CTC GTG ACC GCC GGC GAG Not Xho T7 Promoter AGT GAG TCG TAT TAC AAT TCA TCA CTC AGC ATA AT G TTA AGT Comments for pCR II TOPO 3973 nucleotides LacZa gene bases 1 589 Sp6 Promoter Y ATT ACG CCA AGC TAT TTA GGT GAC ACT ATA GAA TAA TGC GGT TCG ATA AAT CCA CTG TGA TAT CTT
5. 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 Spread 10 50 ul from each transformation on a prewarmed selective plate 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 Incubate plates at 37 C If you are using ampicillin selection visible colonies should appear within 8 hours and blue white screening can be performed after 12 hours For kanamycin selection incubate plates overnight An efficient TOPO Cloning reaction should produce several hundred colonies Pick 10 white or light blue colonies for analysis see Analyzing Positive Clones page 12 Do not pick dark blue colonies Rapid One Shot An alternative protocol is provided below for rapid transformation of One Shot Chemical Mach1 T1 cells This protocol is only recommended for transformations using Transformation ampicillin selection For more information on selecting a transformation Protocol protocol refer to page 6 Note It is essential that LB plates containing ampicillin are prewarmed to 37 C prior to spreading 1 Add 4 ul of the TOPO Cloning reaction from Performing the TOPO Cloning Reaction Step 2 page 5 into a vial of One Shot Chemically Competent E coli and mix gently Do not mix by pipetting up and down Incubate on ice for 5 m
6. Kpn Sac BamH TTG GTA CCG AGC TCG GAT CCA CTA GTA ACG GCC AAC CAT GGC TCG AGC CTA GGT GAT CAT TGC CGG Spe EcoR I EcoR V CTT INAG GGC GAA TTC TGC AGA TAT GAIN PCR Product TTC CCG CTT AAG ACG TCT ATA Nsil Xba Apa EN GAG CAT GCA TCT AGA GGG CCC AAT TCG CCC TAT CTC GTA CGT AGA TCT CCC GGG TTA AGC GGG ATA M13 20 Forward Primer CTG GCC GTC GTT TTA CAA CGT CGT GAC TGG GAA AAC GAC CGG CAG CAA AAT GTT GCA GCA CTG ACC CTT TTG M13 Reverse priming site bases 205 221 Sp6 promoter bases 239 256 Multiple Cloning Site bases 269 383 T7 promoter bases 406 425 M13 20 Forward priming site bases 433 448 f1 origin bases 590 1027 Kanamycin resistance ORF bases 1361 2155 Ampicillin resistance ORF bases 2173 3033 pUC origin bases 3178 3851 Performing the Control Reactions Introduction Before Starting Producing Control PCR Product We recommend performing the following control TOPO Cloning reactions the first 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 the PCR product directly in a TOPO Cloning reaction For each transformation prepare two LB plates containing 50 pg ml kanamycin Note Do not use plates containing ampicillin The control template is a plasmid that encodes ampicillin resistance This template is carried over into
7. TOPO Cloning for the direct insertion of Tag polymerase amplified PCR products into a plasmid vector No ligase post PCR procedures or PCR primers containing specific sequences are required The plasmid vector pCR M TOPO or pCR 2 1 TOPOS is supplied linearized with e Single 3 thymidine T overhangs for TA Cloning e Topoisomerase I covalently bound to the vector referred to as activated vector Tag polymerase has a nontemplate dependent terminal transferase activity that adds a single deoxyadenosine A to the 3 ends of PCR products The linearized vector supplied in this kit has single overhanging 3 deoxythymidine T residues This allows PCR inserts to ligate efficiently with the vector Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites and cleaves the phosphodiester backbone after 5 CCCTT 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 Topoisomerase o og CCCTT PXAGGG GGGAFA PCR Product TTCCC HO 9 SI Topoisomerase continued on next page Overview continued Experimental Outline P
8. proofreading polymerase After purification add Taq polymerase buffer dATP and 0 5 unit of Taq polymerase Incubate the reaction for 10 15 minutes at 72 C and use in the TOPO Cloning reaction Note 22 Recipes LB Luria Bertani Medium and Plates 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 at 15 psi Allow solution to cool to 55 C and add antibiotic if needed 50 pg ml of either ampicillin or kanamycin 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 at 15 psi 3 After autoclaving cool to 55 C add antibiotic 50 ug ml of either ampicillin or kanamycin and pour into 10 cm plates 4 Let harden then invert and store at 4 C in the dark 23 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
9. 3890 bp 3932 bp NsiI 3890 bp 96 3836 bp PstI 1167 2723 bp 1167 2765 bp EcoR I and Afl III 408 693 2789bp 450 693 2789 bp Once the vectors have been adapted with topoisomerase I they are lot qualified using the control reagents included in the kit Under conditions described on pages 17 18 a 750 bp control PCR product was TOPO Cloned into each vector and subsequently transformed into the One Shot competent E coli included with the kit Each lot of vector should yield greater than 95 4 cloning efficiency Both primers have been lot qualified by DNA sequencing experiments using the dideoxy chain termination technique All competent cells are qualified as follows e 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 1 x 10 cfu ug DNA for chemically competent cells and gt 1 x 10 for electrocompetent cells e To verify the absence of phage contamination 0 5 1 ml of competent cells are added to LB top agar and poured onto LB plates After overnight incubation no plaques should be detected e Untransformed cells are plated on LB plates 100 pg ml ampicillin 25 pg ml streptomycin 50 pg ml kanamycin or 15 ug ml chloramphenicol to verify the absence
10. Insert Control PCR Product 1 ql Water 4 ul 3 ul Salt Solution or Dilute Salt 1 ul 1ul Solution TOPO vector 1 ul 1 pl Incubate at room temperature for 5 minutes and place on ice Transform 2 pl of each reaction into separate vials of One Shot competent cells pages 6 10 4 Spread 10 50 ul of each transformation mix onto LB plates containing 50 pg ml kanamycin and X Gal and IPTG if using TOP10F cells Be sure to plate two different volumes to ensure that at least one plate has well spaced colonies For plating small volumes add 20 ul of S O C medium to allow even spreading 5 Incubate overnight at 37 C Hundreds of colonies from the vector PCR insert reaction should be produced 95 4 of these colonies will be white and 90 or more of these will contain the 750 bp insert when analyzed by EcoR I digestion and agarose gel electrophoresis Relatively few colonies will be produced in the vector only reaction and most of these will be dark blue You may observe a few white colonies This results from removal of the 3 deoxythymidine overhangs creating a blunt end vector Ligation re joining of the blunt ends will result in disruption of the LacZa reading frame leading to the production of white colonies pUC19 plasmid is included to check the transformation efficiency of the One Shot competent cells Transform with 10 pg per 50 ul of cells using the protocols on pages 6 10 Use LB plates co
11. Solution in the TOPO Cloning Reaction brings the final concentration of NaCl and MgCl in the TOPO Cloning reaction to 50 mM and 2 5 mM respectively To prevent arcing of your samples during electroporation the volume of cells should be between 50 and 80 pl 0 1 cm cuvettes or 100 to 200 pl 0 2 cm cuvettes If you experience arcing try one of the following suggestions Reduce the voltage normally used to charge your electroporator by 10 Reduce the pulse length by reducing the load resistance to 100 ohms Precipitate the TOPO Cloning reaction and resuspend in water prior to electroporation 11 Analyzing Transformants Analyzing Positive 1 Take 2 6 white or light blue colonies and culture them overnight in Clones Sequencing Analyzing Transformants by PCR 12 LB medium containing 50 ug ml ampicillin or 50 ug ml kanamycin Note If you transformed One Shot Mach1 T1 competent E coli you may inoculate overnight grown colonies and culture them for 4 hours in prewarmed LB medium containing 50 pg ml ampicillin or 50 pg ml kanamycin before isolating plasmid For optimal results we recommend inoculating as much of a single colony as possible 2 Isolate plasmid DNA using PureLink Quick Plasmid Miniprep Kit supplied with cat nos K4500 02 and K4510 02 or available separately page viii The plasmid isolation protocol is included in the manual supplied with the PureLink Quick Plasmid Miniprep Kit and is also
12. available for downloading from www invitrogen com Other kits for plasmid DNA purification are also suitable for use 3 Analyze the plasmids by restriction analysis to confirm the presence and correct orientation of the insert Use a restriction enzyme or a combination of enzymes that cut once in the vector and once in the insert You may sequence your construct to confirm that your gene is cloned in the correct orientation The M13 Forward 20 and M13 Reverse primers are included to help you sequence your insert Refer to the maps on page 15 pCR 2 1 TOPO or page 16 pCR II TOPO for sequence surrounding the TOPO TA Cloning site For the full sequence of either vector refer to our Web site www invitrogen com or contact Technical Service page 24 You may wish to use PCR to directly analyze positive transformants For PCR primers use either the M13 Forward 20 or the M13 Reverse primer and a primer that hybridizes within your insert If you are using this technique for the first time we recommend performing restriction analysis in parallel Artifacts may be obtained because of mispriming or contaminating template The protocol is provided below for your convenience Other protocols are suitable Materials Needed PCR SuperMix High Fidelity Invitrogen Catalog no 10790 020 Appropriate forward and reverse PCR primers 20 uM each Procedure 1 For each sample aliquot 48 ul of PCR SuperMix High Fidelity into a 0 5 m
13. of antibiotic resistant contamination 25 Purchaser Notification Information for European Customers Limited Use Label License No 5 Invitrogen Technology 26 The Mach1 T1 E coli strain is genetically modified to carry the lacZAM15 hsdR lacX74 recA endA tonA genotype As a condition of sale 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 party 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
14. of competent cells and two selective plates e Equilibrate a water bath to 42 C for chemical transformation or set up your electroporator 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 see Important note below e Spread 40 pl of 40 mg ml X gal on each LB plate and incubate at 37 C until ready for use e For TOP10F cells spread 40 pl of 100 mM IPTG in addition to X gal on each LB plate and incubate at 37 C until ready for use IPTG is required for blue white screening e Thaw on ice 1 vial of One Shot cells for each transformation If you are performing the rapid chemical transformation protocol it is essential that you prewarm your LB plates containing 50 100 pg ml ampicillin prior to spreading continued on next page Transforming One Shot DH5a T1 TOP10 and TOP10F Competent Cells continued One Shot Chemical Transformation Protocol Rapid One Shot Chemical Transformation Protocol 10 9x rg qe oso Add 2 ul of the TOPO Cloning reaction from Performing the TOPO Cloning Reaction Step 2 page 5 into a vial of One Shot Chemically Competent E coli and mix gently Do not mix by pipetting up and down Incubate on ice for 5 to 30 minutes Note Longer incubations on ice do not seem to have any affect on transformation efficiency The length of the incubation is at the user s discretion Heat shock the cells for 30 seconds a
15. reaction for 20 to 30 minutes e Concentrate the PCR product Map of pCR 2 1 TOPO pCR 2 1 TOPO Map The map below shows the features of pCR 2 1 TOPO and the sequence surrounding the TOPO Cloning site Restriction sites are labeled to indicate the actual cleavage site The arrow indicates the start of transcription for 17 polymerase The complete sequence of pCR 2 1 TOPO is available for downloading from our Web site www invitrogen com or by contacting Technical Service page 24 lacZa ATG M13 Reverse Primer CAG GAA ACA GCT ATG AC GTC CTT TGT CGA TAC TG ATG ATT ACG TAC TAA TGC GIA BstX EcoRI AGT GTG CTG GAA TTC TCA CAC GAC CTT AAG GTA ACG GCC GCC CAT TGC CGG CGG EcoR V BstX Not Xho AGA TAT CCA TCA CAC TGG CGG CCG CTC TCT ATA GGT AGT GTG ACC GCC GGC GAG T7 Promoter a Ill Kpr I siis I Pank I spe l CCA AGC TTG GTA CCG AGC TCG GAT CCA CTA GGT TCG AAC CAT GGC TCG AGC CTA GGT GAT EcoR GCC CTT AIAG GGC GAA TTC TGC CCG co MM TTC CCG CTT AAG ACC Nsi Xba Apa GAG CAT GCA TCT AGA GGG CTC GTA CGT AGA TCT CCC CCC TAT GGG ATA CCC AAT TCG GGG TTA AGC M13 Forward 20 Primer AAT TCA TTA AGT CTG GCC AGT GAG TCG TAT TA GAC CGG TCA CTC AGC ATA AT GTC GTT TTA CAA CGT CGT CAG CAA AAT GTT GCA GCA GAC TGG GAA AAC CTG ACC CTT TTG pCR 2 1 TOPO 3 9 Comments for pCR 2 1 TOPO 3931 nucleotides
16. vii Accessory Products Additional Products viii The table below lists additional products that may be used with TOPO TA Cloning Kits For more information visit www invitrogen com or contact Technical Service page 24 Item Amount Catalog no Tag DNA Polymerase Native 100 units 18038 018 500 units 18038 042 Tag DNA Polymerase Recombinant 100 units 10342 053 500 units 10342 020 Platinum Tag DNA Polymerase High 100 units 11304 011 Fidelity One Shot TOP10 Chemically Competent 10 reactions C4040 10 E coli 20 reactions C4040 03 40 reactions C4040 06 One Shot TOP10 Electrocompetent 10 reactions C4040 50 E coli 20 reactions C4040 52 One Shot Mach1 T1 Chemically 20 reactions C8620 03 Competent E coli One Shot MAX Efficiency DH5a T1R 20 reactions 12297 016 Chemically Competent E coli One Shot TOP10F Chemically 20 reactions C3030 03 Competent E coli 40 reactions C3030 06 Ampicillin 200 mg 11593 019 Kanamycin 5g 11815 024 25g 11815 032 100 ml 10 mg ml 18160 054 X gal 100 mg 15520 034 1g 15520 018 IPTG 1g 15529 019 S O C Medium 10 x 10 ml 15544 034 PureLink Quick Plasmid Miniprep Kit 50 reactions K2100 10 PureLink Quick Gel Extraction Kit 50 reactions K2100 12 Overview Introduction How It Works Methods TOPO TA Cloning provides a highly efficient 5 minute one step cloning strategy
17. 520 01 40 DH5a T1F chem competent K4520 40 20 TOP10F chem competent K4550 01 40 TOP10F chem competent K4550 40 20 TOP10 electrocompetent K4560 01 40 TOP10 electrocompetent K4560 40 TOPO TA Cloning Kit 20 TOP10 chem competent K4500 02 with pCR 2 1 TOPO and 20 Mach1 T1 chem competent K4510 02 PureLink Quick Plasmid Miniprep Kit TOPO TA Cloning Kit 20 TOP10 chem competent K4600 01 Dual Promoter 40 TOP10 chem competent K4600 40 with pCR IETOPO 20 Mach1 T1 chem competent K4610 20 20 DH5a T1F chem competent K4620 01 40 DH5a T1 chem competent K4620 40 20 TOP10F chem competent K4650 01 40 TOP10F chem competent K4650 40 20 TOP10 electrocompetent K4660 01 40 TOP10 electrocompetent K4660 40 continued on next page Kit Contents and Storage continued TOPO TA Cloning TOPO TA Cloning reagents Box 1 are listed below Note that the user must supply Taq polymerase Store Box 1 at 20 C Reagents Sequence of Primers Item Concentration Amount pCR 2 1 TOPO or 10 ng pl plasmid DNA in 20 ul pCR IL TOPO 50 glycerol 50 mM Tris HCl pH 7 4 at 25 C 1 mM EDTA 1 mM DTT 0 1 Triton X 100 100 ug ml BSA phenol red 10X PCR Buffer 100 mM Tris HCl pH 8 3 at 42 C 100 ul 500 mM KCI 25 mM MgCl 0 01 gelatin Salt Solution 1 2 M NaCl 50 ul 0 06 M MgCl dNTP Mix 12 5 mM dATP 12 5 mM dCTP 10 ul 12 5 m
18. Invitrogen TOPO TA Cloning Five minute cloning of Taq polymerase amplified PCR products Catalog nos K4500 01 K4500 40 K4510 20 K4520 01 K4520 40 K4550 01 K4550 40 K4560 01 K4560 40 K4500 02 K4510 22 pCR 2 1 TOPO Catalog nos K4600 01 K4600 40 K4610 20 K4620 01 K4620 40 K4650 01 K4650 40 K4660 01 K4660 40 pCR II TOPO Version U 10 April 2006 25 0184 ii Table of Contents Kit Contents and Storage ini iii id ada A iv Accessory PO LUCES iii A teet eer e e A e eet ee Ir dee ere EENE vii MethodS ums 1 OVervie A E tem ele dut 1 Producing PER Products inca nate enge Amet seta Ad lus dante 3 Setting Up the TOPO Cloning Reactions xac ertt AA prb de A dint 4 General Guidelines for Transforming Competent Cells sse 6 Transforming One Shot MachT T1 Competent Cells ae tet eU S Ee RR EUR E tue 7 Transforming One Shot DH5a T1 TOP10 and TOP10F Competent Cells sss 9 Analyzing Transformants Sesen oo eE ple ete ther eddie ses ea ie dert ederet toe 12 Optimizing the TOPO Cloning Rescatan te condat f ee seek AL ike 14 Map OF PCR 2 TORO i n 15 Map of pcR WTO OP as saci tes eiin arth ES 16 Performing the Control R actions issiria iii tenete tenente tenete tenens 17 Pise A n 20 Puritving PCR Produets ceste diee tt tee eed eae e NR tetas 20 Addition of 3 A Overhangs Post Amplification cococoninononononnnnncnencncnenncnnnrorannnnnnrnnnaron
19. M dGTP 12 5 mM dTTP neutralized at pH 8 0 in water M13 Forward 20 Primer 0 1 pg pl in TE Buffer 20 ul M13 Reverse Primer 0 1 pg pl in TE Buffer 20 ul Control Template 0 1 pg pl in TE Buffer 10 ul Control PCR Primers 0 1 pg pl each in TE Buffer 10 ul Water 1 ml The table below describes the sequence and pmoles supplied of the sequencing primers included in this kit 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 Genotypes of E coli Strains Information for Non U S Customers Using Mach1 T1 Cells vi The table below describes the items included in each One Shot competent cell kit Store at 80 C 0 5 mM EDTA pH 8 Item Composition Amount S O C Medium 2 Tryptone 6ml may be stored at 4 C or 0 5 Yeast Extract room temperature 10 mM NaCl 25mMKCI 10 mM MgCl 10 mM MgSO 20 mM glucose TOP10 Mach1 T1 DH5a Chemically Competent 21x50 ul T18 or TOP10F cells OR TOP10 cells Electrocomp pUC19 Control DNA 10 pg ul in 5 mM Tris HCI 50 ul DH5a T1 Use this strain for general cloning and blue white screening without IPTG Strain is resistant to T1 bacteriophage F 80lacZAM15 A lacZY A argF U169 recA1 end A1 hsdR17 rc mx phoA supE44 thi 1 gyrA96 relA1 tonA confers resi
20. The information below will help you optimize the TOPO Cloning reaction for your particular needs The high efficiency of TOPO Cloning technology allows you to streamline the cloning process If you routinely clone PCR products and wish to speed up the process consider the following e Incubate the TOPO Cloning reaction for only 30 seconds instead of 5 minutes You may not obtain the highest number of colonies but with the high efficiency of TOPO Cloning most of the transformants will contain your insert e After adding 2 ul of the TOPO Cloning reaction to chemically competent cells incubate on ice for only 5 minutes Increasing the incubation time to 30 minutes does not significantly improve transformation efficiency If you are TOPO Cloning large PCR products toxic genes or cloning a pool of PCR products you may need more transformants to obtain the clones you want To increase the number of colonies e Incubate the salt supplemented TOPO Cloning reaction for 20 to 30 minutes instead of 5 minutes Increasing the incubation time of the salt supplemented TOPO Cloning reaction allows more molecules to ligate increasing the transformation efficiency Addition of salt appears to prevent topoisomerase from rebinding and nicking the DNA after it has ligated the PCR product and dissociated from the DNA To clone dilute PCR products you may e Increase the amount of the PCR product e Incubate the TOPO Cloning
21. ananannnnararananarornrnnnnnanos 22 RECT POS ee ooa eno E ME do A trib tu E ed 23 Technical Service x5 aede pde eed a E ep dep b ne A 24 Product Qualifications i siete qu Ee ee ese b n petet e e dde 25 Purchaser Notification nente edades elit denen 26 References oe b eth en ER A eese AA AAA T e ec eet eed 27 iii Kit Contents and Storage Shipping and Storage Types of TOPO TA TOPO TA Cloning Kits are shipped on dry ice Each kit contains a box with TOPO TA Cloning reagents Box 1 and a box with One Shot Chemically Competent or Electrocomp cells Box 2 TOPO TA Cloning Kits supplied with the PureLink Quick Plasmid Miniprep Kit cat nos K4500 02 and K4510 02 are shipped with an additional box containing reagents for plasmid purification Box 3 Store Box 1 at 20 C Box 2 at 80 C and Box 3 at room temperature TOPO TA Cloning Kits are available with pCR 2 1 TOPO or pCR II TOPO Cloning Kits vector and a choice of One Shot Chemically or Electrocomp Competent cells as described in the table below Select TOPO TA Cloning Kits are also available with PureLink Quick Plasmid Miniprep Kit See page vi for the genotypes of the strains Product Reactions One Shot Cells Type of Cells Catalog no TOPO TA Cloning Kit 20 TOP10 chem competent K4500 01 with pCR 2 1 TOPOS 40 TOP10 chem competent K4500 40 20 Mach1 T1 chem competent K4510 20 20 DH5a T1F chem competent K4
22. and transformation reactions resulting in transformants that are ampicillin resistant and white but are not the desired construct Transforming One Shot Mach1 T1 Competent Cells Introduction Note User Supplied Materials Preparing for Transformation Important Protocols to transform One Shot Mach1 T1 chemically competent E coli are provided below If are transforming cells other than Mach1 T1 cells refer to the section entitled Transforming One Shot DH5a T1 TOP10 and TOP10F Competent Cells pages 9 11 The Mach1 T1 strain allows you to visualize colonies 8 hours after plating on ampicillin selective plates If you are using kanamycin selection you will need to incubate plates overnight in order to visualize colonies With the Mach1 TI strain you may also prepare plasmid DNA 4 hours after inoculating a single overnight grown colony Note that you will get sufficient growth of transformed cells within 4 hours in either ampicillin or kanamycin selective media In addition to general microbiological supplies e g plates spreaders you will need the following reagents and equipment e TOPO Cloning reaction from Performing the TOPO Cloning Reaction Step 2 page 5 e S O C medium included with the kit e LB plates containing 50 ug ml ampicillin or 50 pg ml kanamycin e 40 mg ml X gal in dimethylformamide DMF e 42 C water bath e 37 C shaking and non shaking incubator
23. 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 invitrogen com 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 Mol
24. d with your kit General guidelines for transformation are provided below For transformation protocols refer to the section entitled Transforming One Shot Mach1 T1 Competent Cells pages 7 8 or Transforming One Shot DH5a T1 TOP10 and TOP10F Competent Cells pages 9 11 depending on the competent EF coli you wish to transform Two protocols are provided to transform One Shot chemically competent E coli Consider the following factors when choosing the protocol that best suits your needs If you wish to Then use the maximize the number of transformants regular chemical transformation clone large PCR products 21000 bp protocol use kanamycin as the selective agent see Important note below rapid chemical transformation protocol obtain transformants as quickly as possible If you will be using kanamycin as the selective agent for chemical transformation use the regular chemical transformation protocol The rapid chemical transformation protocol is only suitable for transformations using ampicillin selection If you use a plasmid template for your PCR that carries either the ampicillin or kanamycin resistance marker we recommend that you use the other selection agent to select for transformants For example if the plasmid template contains the ampicillin resistance marker then use kanamycin to select for transformants The template is carried over into the TOPO Cloning
25. ducts Catalog no K2700 20 PCR cloning artifacts false positives TOPO Cloning is very efficient for small fragments 100 bp present in certain PCR reactions Gel purify your PCR product page 20 PCR product does not contain sufficient 3 A overhangs even though you used Tag polymerase Increase the final extension time to ensure all 3 ends are adenylated Tag polymerase is less efficient at adding a nontemplate 3 A next to another A Taq is most efficient at adding a nontemplate 3 A next to a C You may have to redesign your primers so that they contain a 5 G instead of a 5 T Brownstein et al 1996 19 Appendix Purifying PCR Products Introduction Smearing multiple banding primer dimer artifacts or large PCR products gt 1 kb may necessitate gel purification If you intend 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 Two simple protocols are described in this section TM Using the The PureLink Quick Gel Extraction Kit page viii allows you to rapidly purify PureLink Quick PCR products from regular agarose gels Gel Extraction Kit 1 2 10 11 12 13 14 Equilibrate a water bath or heat block to 50 C
26. ecular Biology New York Greene Publishing Associates and Wiley Interscience Brownstein M J Carpten J D and Smith J R 1996 Modulation of Non Templated Nucleotide Addition by Taq DNA Polymerase Primer Modifications that Facilitate Genotyping BioTechniques 20 1004 1010 Innis M A Gelfand D H Sninsky J J and White T S 1990 PCR Protocols A Guide to Methods and Applications Academic Press San Diego CA Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Edition Plainview New York Cold Spring Harbor Laboratory Press Shuman S 1991 Recombination Mediated by Vaccinia Virus DNA Topoisomerase I in Escherichia coli is Sequence Specific Proc Natl Acad Sci USA 88 10104 10108 Shuman S 1994 Novel Approach to Molecular Cloning and Polynucleotide Synthesis Using Vaccinia DNA Topoisomerase J Biol Chem 269 32678 32684 1999 2006 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 27
27. ells see below For this reason two different TOPO Cloning reactions are provided to help you obtain the best possible results Read the following information carefully For TOPO Cloning and transformation into chemically competent E coli adding sodium chloride and magnesium chloride to a final concentration of 200 mM NaCl 10 mM MgCl in the TOPO Cloning reaction increases the number of colonies over time A Salt Solution 1 2 M NaCl 0 06 M MgCl is provided to adjust the TOPO Cloning reaction to the recommended concentration of NaCl and MgCl For TOPO Cloning and transformation of electrocompetent E coli salt must also be included in the TOPO Cloning reaction but the amount of salt must be reduced to 50 mM NaCl 2 5 mM MgCl to prevent arcing The Salt Solution is diluted 4 fold to prepare a 300 mM NaCl 15 mM MgCl solution for convenient addition to the TOPO Cloning reaction see next page continued on next page Setting Up the TOPO Cloning Reaction continued Setting Up the The table below describes how to set up your TOPO Cloning reaction 6 ul for TOPO Cloning eventual transformation into either chemically competent or electrocompetent Reaction TOP10 or chemically competent DH5a T1 Mach1 T1 or TOP10F One Shot E coli Additional information on optimizing the TOPO Cloning reaction for your needs can be found on page 14 Note The red color of the TOPO vector solution is normal and i
28. emperature for 5 minutes Centrifuge at gt 12 000 x g for 1 minute Discard flow through Centrifuge the column at gt 12 000 x g for 1 minute to remove any residual buffer Place the column into a 1 5 ml Recovery Tube Add 50 ul warm 65 70 C TE Buffer TE to the center of the cartridge Incubate at room temperature for 1 minute Centrifuge at 212 000 x g for 2 minutes The Recovery Tube contains the purified DNA Store DNA at 20 C Discard the column Use 4 ul of the purified DNA for the TOPO Cloning reaction 20 continued on next page Purifying PCR Products continued Low Melt Agarose Note that gel purification will result in a dilution of your PCR product Use only chemically competent cells for transformation Method Note 1 Electrophorese all of your PCR reaction on a low melt TAE agarose gel 0 8 to 1 2 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 Use 4 pl of the melted agarose containing your PCR product in the TOPO Cloning reaction page 5 Incubate the TOPO Cloning reaction at 37 C for 5 to 10 minutes This is to keep the agarose melted Transform 2 to 4 pl directly into competent One Shot cells using one of the methods described on pages 10 11 Note that the cloning efficiency may decrease with purification of the PCR
29. everal hundred colonies Pick 10 white or light blue colonies for analysis see Analyzing Positive Clones page 12 Do not pick dark blue colonies continued on next page Transforming One Shot DH5a T1 TOP10 and TOP10F Competent Cells continued One Shot Electroporation Protocol Note Add 2 ul of the TOPO Cloning reaction from Performing the TOPO Cloning Reaction Step 2 page 5 into a vial of One Shot Electrocompetent E coli and mix gently Do not mix by pipetting up and down Carefully transfer solution to a 0 1 cm cuvette to avoid formation of bubbles Electroporate your samples using your own protocol and your electroporator Note If you have problems with arcing see below Immediately add 250 ul of room temperature S O C medium Transfer the solution to a 15 ml snap cap tube e g Falcon and shake for at least 1 hour at 37 C to allow expression of the antibiotic resistance genes Spread 10 50 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 An efficient TOPO Cloning reaction should produce several hundred colonies Pick 10 white or light blue colonies for analysis see Analyzing Positive Clones next page Do not pick dark blue colonies Addition of the Dilute Salt
30. if you are using genomic DNA as a template Use the cycling parameters suitable for your primers and template Be sure to include a 7 to 30 minute extension at 72 C after the last cycle to ensure that all PCR products are full length and 3 adenylated DNA Template 10 100 ng 10X PCR Buffer 5 ul 50 mM dNTPs 0 5 ul Primers 100 200 ng each 1 uM each Water add to a final volume of 49 pl Tag Polymerase 1 unit ul 1 ul Total Volume 50 ul 2 Check the PCR product by agarose gel electrophoresis You should see a single discrete band If you do not see a single band refer to the Note below If you do not obtain a single discrete band from your PCR you may gel purify your fragment before using the TOPO TA Cloning Kit see page 20 Take special care to avoid sources of nuclease contamination Alternatively you may optimize your PCR to eliminate multiple bands and smearing Innis et al 1990 The PCR Optimizer Kit Catalog no K1220 01 incorporates many of the recommendations found in this reference For more information refer to our Web site www invitrogen com or contact Technical Service page 24 Setting Up the TOPO Cloning Reaction Introduction Note Important Transforming Chemically Competent E coli Transforming Electrocompetent E coli Once you have produced the desired PCR product you are ready to TOPO Clone it into the pCR 2 1 TOPO or pCR II TOPO vector and transform the recombinant vecto
31. inutes Spread 50 ul of cells on a prewarmed LB plate containing 50 100 pg ml ampicillin and incubate overnight at 37 C An efficient TOPO Cloning reaction should produce several hundred colonies Pick 10 white or light blue colonies for analysis see Analyzing Positive Clones page 12 Do not pick dark blue colonies Transforming One Shot DH5a T1 TOP10 and TOP10F Competent Cells Introduction Materials Supplied by the User Preparation for Transformation Important Protocols to transform One Shot DH5a T1 TOP10 and TOP10F competent E coli are provided below Both chemical transformation and electroporation protocols are provided If you are transforming Mach1 T1 cells refer to the section entitled Transforming One Shot Mach1 T1 Competent Cells pages 7 8 In addition to general microbiological supplies e g plates spreaders you will need the following reagents and equipment e TOPO Cloning reaction from Performing the TOPO Cloning Reaction Step 2 page 5 e SOC medium included with the kit e LB plates containing 50 ug ml ampicillin or 50 pg ml kanamycin e 40 mg ml X gal in dimethylformamide DMF e 100 mM IPTG in water for use with TOP10F e 15 mlsnap cap plastic culture tubes sterile electroporation only e 42 C water bath or an electroporator and 0 1 or 0 2 cm cuvettes e 37 C shaking and non shaking incubator For each transformation you will need one vial
32. l microcentrifuge tube Add 1 ul each of the forward and reverse PCR primer 2 Pick 10 colonies and resuspend them individually in 50 ul of the PCR cocktail from Step 1 above Don t forget to make a patch plate to preserve the colonies for further analysis 3 Incubate the reaction for 10 minutes at 94 C to lyse the 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 Visualize by agarose gel electrophoresis continued on next page Analyzing Transformants continued If you have problems obtaining transformants or the correct insert perform the Important control reactions described on pages 17 18 These reactions will help you troubleshoot your experiment Long Term Once you have identified the correct clone be sure to prepare a glycerol stock for Storage long term storage We recommend that you store a stock of plasmid DNA at 20 C 1 Streak the original colony out on LB plates containing 50 pg ml ampicillin or 50 ug ml kanamycin 2 Isolate a single colony and inoculate into 1 2 ml of LB containing 50 pg ml ampicillin or 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 Store at 80 C 13 Optimizing the TOPO Cloning Reaction Introduction Faster Subcloning More Transformants Cloning Dilute PCR Products 14
33. ntaining 100 pg ml ampicillin Just before plating the transformation mix for electrocompetent cells dilute 10 ul of the mix with 90 pl of S O C medium Type of Cells Volume to Plate Transformation Efficiency Chemically Competent 10 ul 20 ul S O C 1 x10 cfu ug DNA Electrocompetent 20 ul 1 10 dilution gt 1x10 cfu ug DNA continued on next page Performing the Control Reactions continued Factors Affecting Cloning Efficiency Note that lower cloning efficiencies will result from the following variables Most of these are easily correctable but if you are cloning large inserts you may not obtain the expected 95 4 cloning efficiency Variable Solution pH gt 9 Check the pH of the PCR amplification reaction and adjust with 1 M Tris HCI pH 8 Incomplete extension during PCR Be sure to include a final extension step of 7 to 30 minutes during PCR Longer PCR products will need a longer extension time Cloning large inserts gt 1 kb Try one or all of the following Increase amount of insert Incubate the TOPO Cloning reaction longer Gel purify the insert see page 20 Excess or overly dilute PCR product Reduce or concentrate the amount of PCR product Cloning blunt ended fragments Add 3 A overhangs to your blunt PCR product by incubating with Tag polymerase page 22 Use the Zero Blunt PCR Cloning Kit to clone blunt PCR pro
34. product You may wish to optimize your PCR to produce a single band 21 Addition of 3 A Overhangs Post Amplification Introduction Direct cloning of DNA amplified by proofreading polymerases into TOPO TA Cloning vectors is often difficult because proofreading polymerases remove the 3 A overhangs necessary for TA Cloning Invitrogen has developed a simple method to clone these blunt ended fragments Before Starting You will need the following items e Taq polymerase e A heat block equilibrated to 72 C e Phenol chloroform optional e 3 M sodium acetate optional e 100 ethanol optional e 80 ethanol optional e TE buffer optional Procedure This is just one method for adding 3 adenines Other protocols may be suitable 1 After amplification with a proofreading polymerase place vials on ice and add 0 7 1 unit of Tag polymerase per tube Mix well It is not necessary to change the buffer A sufficient number of PCR products will retain the 3 A overhangs 2 Incubate at 72 C for 8 10 minutes do not cycle 3 Place on ice and use immediately in the TOPO Cloning reaction Note If you plan to store your sample overnight before proceeding with TOPO Cloning extract your sample with an equal volume of phenol chloroform to remove the polymerases Ethanol precipitate the DNA and resuspend in TE buffer using the starting volume of the PCR You may also gel purify your PCR product after amplification with a
35. r into competent E coli It is important to 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 sections detailing transformation of competent cells pages 6 10 before beginning If this is the first time you have TOPO Cloned perform the control reactions on pages 17 18 in parallel with your samples Recent experiments at Invitrogen demonstrate that inclusion of salt 200 mM NaCl 10 mM MgCl in the TOPO Cloning reaction increases the number of transformants 2 to 3 fold We have also observed that in the presence of salt incubation times of greater than 5 minutes can also increase the number of transformants This is in contrast to earlier experiments without salt where the number of transformants decreases as the incubation time increases beyond 5 minutes Inclusion of salt allows for longer incubation times because it prevents topoisomerase I from rebinding and potentially nicking the DNA after ligating the PCR product and dissociating from the DNA The result is more intact molecules leading to higher transformation efficiencies Because of the above results we recommend adding salt to the TOPO Cloning reaction A stock salt solution is provided in the kit for this purpose Note that the amount of salt added to the TOPO Cloning reaction varies depending on whether you plan to transform chemically competent cells or electrocompetent c
36. roduce Your PCR Product Set Up TOPO Cloning Reaction Mix Together PCR Product and TOPO Vector Incubate 5 Minutes at Room Temperature Transform TOPO Cloning Reaction into One Shot Competent Cells Select and Analyze 10 White or Light Blue Colonies for Insert Producing PCR Products Introduction Note Materials Supplied by the User Polymerase Mixtures Producing PCR Products Note It is important to properly design your PCR primers to ensure that you obtain the product you need for your studies Once you have decided on a PCR strategy and have synthesized the primers you are ready to produce your PCR product Remember that your PCR product will have single 3 adenine overhangs Do not add 5 phosphates to your primers for PCR The PCR product synthesized will not ligate into pCR 2 1 TOPO or pCR II TOPO You will need the following reagents and equipment e Taq polymerase e Thermocycler e DNA template and primers for PCR product If you wish to use a mixture containing Taq polymerase and a proofreading polymerase Taq must be used in excess of a 10 1 ratio to ensure the presence of 3 A overhangs on the PCR product If you use polymerase mixtures that do not have enough Taq polymerase or a proofreading polymerase only you can add 3 A overhangs using the method on page 22 1 Set up the following 50 ul PCR reaction Use less DNA if you are using plasmid DNA as a template and more DNA
37. s used to visualize the solution Reagent Chemically Competent E coli Electrocompetent E coli Fresh PCR product 0 5 to 4 pl 0 5 to4 ul Salt Solution 1 ul Dilute Salt Solution 1 ul Water add to a total volume of 5 pl add to a total volume of 5 pl TOPO vector 1 ul 1 ul Final Volume 6 ul 6 ul Store all reagents at 20 C when finished Salt solutions and water can be stored at room temperature or 4 C Performing the 1 Mix reaction gently and incubate for 5 minutes at room temperature 22 23 C TOPO Cloning Note For most applications 5 minutes will yield plenty of colonies for Reaction 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 1 kb or if you are TOPO Cloning a pool of PCR products increasing the reaction time will yield more colonies 2 Place the reaction on ice and proceed to General Guidelines for Transforming Competent Cells next page Note You may store the TOPO Cloning reaction at 20 C overnight General Guidelines for Transforming Competent Cells Introduction Selecting a One Shot Chemical Transformation Protocol Important Once you have performed the TOPO Cloning reaction you will transform your pCR 2 1 TOPO or pCR H TOPO construct into the competent E coli provide
38. stance to phage T1 Mach1 T1 Use this strain for general cloning and blue white screening without IPTG Strain is resistant to T1 bacteriophage F 80 lacZ AM15 AlacX74 hsdR rx mx ArecA1398 end A1 ton A confers resistance to phage T1 TOP10 Use this strain for general cloning and blue white screening without IPTG F mcr A A mrr hsdRMS mcrBC 80lacZAM15 AlacX74 recA1 araD139 A ara leu 7697 galU galK rpsL Str endA1 nupG TOP10F This strain overexpresses the Lac repressor lacI3 gene For blue white screening you will need to add IPTG to the plates to obtain expression from the lac promoter This strain contains the F episome and can be used for single strand rescue of plasmid DNA containing an f1 origin F lacla Tn10 Tet mcrA A mrr hsdRMS mcrBC 80lacZAM15 AlacX74 recA1 araD139 A ara leu 7697 galU galK rpsL Str endA1 nupG The parental strain of Mach1 T1 E 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 continued on next page Kit Contents and Storage continued PureLink Quick For kit components of the PureLink Quick Plasmid Miniprep Kit Box 3 Plasmid Miniprep supplied with cat nos K4510 02 and K4500 02 refer to the manual supplied with Kit the miniprep kit
39. t 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 Spread 10 50 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 An efficient TOPO Cloning reaction should produce several hundred colonies Pick 10 white or light blue colonies for analysis see Analyzing Positive Clones page 12 Do not pick dark blue colonies An alternative protocol is provided below for rapid transformation of One Shot chemically competent E coli This protocol is only recommended for transformations using ampicillin selection For more information on selecting a transformation protocol refer to page 6 Note It is essential that LB plates containing ampicillin are prewarmed prior to spreading 1 Add 4 ul of the TOPO Cloning reaction from Performing the TOPO Cloning Reaction Step 2 page 5 into a vial of One Shot Chemically Competent E coli and mix gently Do not mix by pipetting up and down Incubate on ice for 5 minutes Spread 50 pl of cells on a prewarmed LB plate containing 50 100 ug ml ampicillin and incubate overnight at 37 C An efficient TOPO Cloning reaction should produce s
40. the TOPO Cloning and transformation reactions Transformants carrying this plasmid will also be ampicillin resistant and white resulting in an apparent increase in TOPO Cloning efficiency but upon analysis colonies do not contain the desired construct 1 To produce the 750 bp control PCR product set up the following 50 pl PCR Control DNA Template 100 ng 10X PCR Buffer dNTP Mix Control PCR Primers 0 1 ug ul each Water Tag Polymerase 1 unit ul lul 5yl 0 5 ul 1 ul 41 5 ul 1 ul Total Volume Overlay with 70 ul 1 drop of mineral oil 50 ul Amplify using the following cycling parameters Step Time Temperature Cycles Initial Denaturation 2 minute 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 A discrete 750 bp band should be visible Proceed to the Control TOPO Cloning Reactions next page continued on next page 17 Performing the Control Reactions continued Control TOPO Using the control PCR product produced on the previous page and the TOPO Cloning Reactions vector set up two 6 pl TOPO Cloning reactions as described below Analysis of Results Transformation Control 18 1 Setup control TOPO Cloning reactions Reagent Vector Only Vector PCR
41. 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 or implied including any warranty of merchantability or fitness for a particular purpose Product Qualification Restriction Digest TOPO Cloning Efficiency Primers One Shot Competent E coli Supercoiled pCR 2 1 TOPO and pCR II TOPOC are qualified by restriction digest The table below lists the restriction enzymes and the expected fragments Restriction Enzyme pCR 2 1 TOPO pCR II TOPO Hind III linearizes 3890 bp 3932 bp Xba 1 linearizes
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