TOPO® TA Cloning® Kit User Guide (Pub. no MAN0000047, Rev A.0)
Contents
1. Cloning Kits vector Kits with competent cells are available with One Shot Chemically or Electrocomp competent cells as described in the following table see page 8 for the genotypes of the strains Note Cat no 450641 is not supplied with competent cells Select TOPO TA Cloning Kits are also available with PureLink Quick Plasmid Miniprep Kit Product Cat no One Shot Cells Type of Cells Reactions TOPO TA Cloning Kit K4500 01 TOP10 chem competent 25 with pCR 2 1 TOPO vector K4500 40 TOP10 chem competent 50 K4500 J10 TOP10 chem competent 10 K4510 20 Mach1 T1F chem competent 25 K4520 01 DH5a T1 chem competent 25 K4520 40 DH5a T1 chem competent 50 K4550 01 TOP10F chem competent 25 K4550 40 TOP10F chem competent 50 K4560 01 TOP10 electrocompetent 25 K4560 40 TOP10 electrocompetent 50 450641 Not supplied NA 25 TOPO TA Cloning Kit K4500 02 TOP10 chem competent 25 with pCR 2 1 TOPO vector 4510 02 Mach1 T1 chem competent 25 and PureLink Quick Plasmid Miniprep Kit TOPO TA Cloning Kit K4600 01 TOP10 chem competent 25 Dual Promoter K4600 40 TOP10 chem competent 50 With per JETP vectori cagao TOP10 chem competent 10 K4610 20 Mach1 T1F chem competent 25 K4620 01 DH5a T1 chem competent 25 K4620 40 DH5a T1 chem competent 50 K4650 01 TOP10F chem competent2. Produce PCR products Introduction It is important to properly design your PCR primers to ensure that you obtain the product you need for your studies After deciding on a PCR strategy and synthesizing the primers you are ready to produce your PCR product Remember that your PCR product will have single 3 adenine overhangs Note Do not add 5 phosphates to your primers for PCR The PCR product synthesized will not ligate into pCR 2 1 TOPO vector or pCR II TOPO vector Materials supplied Taq polymerase by the user e Thermocycler e DNA template and primers for PCR product Polymerase If you wish to use a mixture containing Taq polymerase and a proofreading polymerase Tag must be used in excess of a 10 1 ratio to ensure the presence of 3 A overhangs on the PCR product mixtures If you use polymerase mixtures that do not have enough Taq polymerase or a proofreading polymerase only add 3 A overhangs using the method on page 27 Produce PCR 1 Set up the following 50 uL PCR reaction Use less DNA if you are using plasmid DNA as a template and more DNA 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 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 pL Primers 100 200 ng each 1 uM each Water add t
3. 25 K4650 40 TOP10F chem competent 50 K4660 01 TOP10 electrocompetent 25 K4660 40 TOP10 electrocompetent 50 Continued on next page Contents and storage continued TOPO TA Cloning TOPO TA Cloning reagents Box 1 are listed in the following table Note that reagents the user must supply Taq polymerase Store Box 1 at 30 C to 10 C Amount Item Concentration 10 Rxns 25 Rxns 50 Rxns pCR 2 1 TOPO vector 10 ng L plasmid DNA in 10 uL 25 uL 2x 25 uL or 50 glycerol pCR Il TOPO vector 50 mM Tris HCl pH 7 4 at 25 C 1 mM EDTA 1 mM DTT 0 1 Triton X 100 100 pg mL BSA phenol red 10X PCR Buffer 100 mM Tris HCl pH 8 3 at 42 C 100 pL 100 pL 2 x 100 uL 500 mM KCL 25 mM MgCl 0 01 gelatin Salt Solution 1 2 M NaCl 50 uL 50 uL 2 x 50 uL 0 06 M MgCl dNTP Mix 12 5 mM dATP 12 5 mM dCTP 10 uL 10 uL 2x 10 pL 12 5 mM dGTP 12 5 mM dTTP neutralized at pH 8 0 in water M13 Forward 20 Primer 0 1 ug L in TE Buffer 20 uL 20 uL 2x 20 uL M13 Reverse Primer 0 1 ug uL in TE Buffer 20 uL 20 uL 2x 20 uL Control Template 0 1 ug pL in TE Buffer 10 uL 10 uL 2x 10 pL Control PCR Primers 0 1 ug uL each in TE Buffer 10 uL 10 uL 2x 10 pL Water 1 mL 1 mL 2x 1mL Sequence of The following table describes the sequence and pmoles supplied of the primers sequencing primers included in this kit Primer Sequence pMoles Supplied M13
4. Box 2 to room temperature e Warm selective plates at 37 C for 30 minutes see the following Important Note e Spread 40 pL 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 IMPORTANT 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 pre warm your LB plates containing 50 100 pg mL ampicillin prior to spreading Continued on next page Transform One Shot Mach1 T1 competent cells continued One Shot chemical For optimal growth of Mach1 T1 E coli cells it is essential that selective plates transformation are prewarmed to 37 C prior to spreading protocol 1 Dy OU ES Add 2 uL of the TOPO Cloning reaction from Perform the TOPO Cloning reaction step 2 on page 12 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 30 minutes Note Longer incubations on ice do not seem to affect 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 pL 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 transformat
5. TCA CTG GCC GTC GIT TTA CAA CGT CGT GAC TGG GAA AAC TCA CTC AGC ATA ATG TTA AGT GAC CGG CAG CAA AAT GIT GCA GCA CTG ACC CIT TIG Comments for pCR 2 1 TOPO 3931 nucleotides 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 29 Map of pCR Il TOPO pCR Il TOPO map 30 The following map shows the features of the pCR I TOPO vector 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 sequence of the pCR II TOPO vector is available from www lifetechnologies com support or by contacting Technical Support page 32 lacZa ATG M13 Reverse Primer CAG GAA ACA GCT ATG ACC ATG GTC CTT TGT CGA TAC TGG TAC Nsil delta Ml TAC TCA AGC TAT GCA TCA AGC ATG AGT TCG ATA CGT AGT TCG ae FRR GCC AGT GTG CTG GAA TTC GCC CGG TCA CAC GAC CTT AAG CGG BsiX Not Xho CCA TCA CAC TGG CGG CCG CTC GGT AGT GTG ACC GCC GGC GAG T7 Promoter AGT GAG TCG TAT T AAT TCA TCA CTC AGC ATA ATG TTA AGT Comments for pCR Il TOPO 3973 nucleotides LacZa
6. be visible Proceed to the Control TOPO Cloning reactions on page 23 Continued on next page Perform the control reactions continued Control TOPO Cloning reactions Analyze results Transformation control Using the control PCR product produced on page 22 and the TOPO vector set up two 6 pL TOPO Cloning reactions as described below 1 Set up control TOPO Cloning reactions Reagent Vector Only Vector PCR Insert Control PCR Product 1 uL Water 4 uL 3 uL Salt Solution 1 uL 1 uL TOPO vector Tul Tul Final Volume 6 uL 6 uL Incubate the reactions at room temperature for 5 minutes and place on ice Prepare the samples for transformation e For chemical transformation protocols proceed directly to step 4 e For electroporation protocols only dilute the TOPO Cloning reaction 4 fold e g add 18 uL of water to the 6 uL TOPO Cloning reaction before proceeding to step 4 4 Transform 2 pL of each reaction into separate vials of One Shot competent cells pages 13 18 or equivalent competent cells 5 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 pL of S O C medium to allow even spreading 6 Incubate overnight at 37 C Hundreds of colonies
7. 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 Kits containing competent cells include pUC19 plasmid to check the transformation efficiency of the One Shot competent cells Transform with 10 pg per 50 pL of cells using the protocols on pages 13 18 Use LB plates containing 100 pg mL ampicillin Just before plating the transformation mix for electrocompetent cells dilute 10 uL of the mix with 90 uL S O C medium Type of Cells Volume to Plate Transformation Efficiency Chemically competent 10 uL 20 pLS 0 C 1 x 10 cfu ug DNA Electrocompetent 20 uL 1 10 dilution gt 1 x 10 cfu ug DNA Continued on next page 23 Perform the control reactions continued Factors affecting cloning efficiency 24 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 m
8. gene bases 1 589 Sp6 Promoter ATT ACG CCA AGC TAT TTA GGT GAC ACT ATA TAA TGC GGT TCG ATA AAT CCA CTG TGA TAT CIT Kpn eal rd l Spe TTG GTA CCG AGC TCG GAT CCA CTA GTA ACG GCC AAC CAT GGC TCG AGC CTA GGT GAT CAT TGC CGG FENS EPEY CIT AG GGC GAA TTC TGC AGA TAT GAR PCR Product TTC CCG CTT AAG ACG TCT ATA Nsi Xba Apa e 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 Q CGT CGT GAC TGG GAA AAC GAC CGG CAG CAA AAT GTI 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 Appendix C Ordering information Additional products The following table lists additional products that may be used with TOPO TA Cloning Kits For more information visit www lifetechnologies com or contact Technical Support page 32 Item Quantity Cat 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 7ag DNA Polymerase High 100 units 11304 011 Fidelity PCR
9. tube for an additional 5 minutes 6 Preheat an aliquot of TE Buffer TE to 65 70 C 7 Place a Quick Gel Extraction Column into a Wash Tube Pipet the mixture from step 5 of this procedure onto the column Use 1 column per 400 mg agarose 8 Centrifuge at gt 12 000 x g for 1 minute Discard the flow through Place the column back into the Wash Tube 9 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 10 Add 700 pL 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 temperature for 5 minutes Centrifuge at gt 12 000 x g for 1 minute Discard flow through 11 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 12 Add 50 uL warm 65 70 C TE Buffer TE to the center of the cartridge Incubate at room temperature for 1 minute 13 Centrifuge at gt 12 000 x g for 2 minutes The Recovery Tube contains the purified DNA Store DNA at 20 C Discard the column 14 Use 4 pL of the purified DNA for the TOPO Cloning reaction Continued on next page 25 Purify PCR products continued Low melt agarose method Note 26 Note that gel purification will dilute your PCR prod
10. 2 We recommend performing the following control TOPO Cloning reactions the first time you use the kit to help you evaluate 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 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 uL PCR Control DNA Template 100 ng 1 pL 10X PCR Buffer 5 pL dNTP Mix 0 5 uL Control PCR Primers 0 1 ug uL each 1 pL Water 41 5 uL Tag Polymerase 1 unit pL 1 pL Total Volume 50 pL 2 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 1256 Final extension 7 minutes 72 C 1X 3 Remove 10 pL from the reaction and analyze by agarose gel electrophoresis A discrete 750 bp band should
11. 2 to room temperature e Warm selective plates at 37 C for 30 minutes see Important page 17 e Spread 40 uL 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 uL 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 Continued on next page Transform One Shot DH5a T1 TOP10 and TOP10F competent cells continued IMPORTANT One Shot chemical transformation protocol Rapid One Shot chemical transformation protocol If you are performing the rapid chemical transformation protocol it is essential that you prewarm your LB plates containing 50 100 ug ml ampicillin prior to spreading DS OT a9 Add 2 uL of the TOPO Cloning reaction from Perform the TOPO Cloning reaction step 2 on page 12 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 30 minutes Note Longer incubations on ice do not seem to affect 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 pL of room temperature S O C medium Cap the tube tightly and shake the tube horizontally 200 rpm at 37 C for 1 hou
12. AA A a AA aaa EETA E 34 Referentes mitico ed dd E S 35 IMPORTANT Changes from previous version About this guide Before using this product read and understand the information in the Safety appendix in this document Revision Date Description A 0 24 February 2014 e Increase from 20 to 25 reaction kit size e Include Cat nos K4500 J10 K4600 J10 e Version numbering changed to alphanumeric format and reset to A in conformance with internal document control procedures Product information Contents and storage Shipping and storage TOPO TA Cloning Kits are shipped on dry ice Kits containing competent cells contain 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 TOPO TA Cloning Kit for Subcloning Cat no 450641 is shipped with only the TOPO TA Cloning reagents Box 1 Box Store at 1 30 C to 10 C in a non frost free freezer 85 C to 68 C Room temperature 15 C to 30 C Continued on next page Contents and storage continued Types of TOPO TA TOPO TA Cloning Kits are available with pCR 2 1 TOPO or pCR II TOPO
13. Forward 20 5 GTAAAACGACGGCCAG 3 407 M13 Reverse 5 CAGGAAACAGCTATGAC 3 385 PureLink Quick Plasmid Miniprep Kit For kit components of the PureLink Quick Plasmid Miniprep Kit Box 3 supplied with Cat nos K4510 02 and K4500 02 refer to the manual supplied with the miniprep kit Continued on next page Contents and storage continued One Shot reagents The following table describes the items included in each One Shot competent cells kit Store at 85 C to 68 C Amount Item Composition 10 Rxns 25 Rxns 50 Rxns S 0 C Medium 2 Tryptone 6mL 6mL 2x6mL may be stored at 4 C 0 5 Yeast Extract or room temperature 10 mM NaCl 2 5 mM KCL 10 mM MgClo 10 mM MgSO 20 mM glucose TOP10 Mach1 T1F Chemically competent 11x 50 uL 26x 50uL 2x 26x 50 uL DH5a T1 or TOP10F or TOP10 cells Electrocomp pUC19 Control DNA 10 pg pL 50 uL 50 uL 2x 50 uL Genotypes of E coli strains DH5a T1 Use this strain for general cloning and blue white screening without IPTG Strain is resistant to T1 bacteriophage F 480lacZAM15 A lacZY A argF U169 recA1 endA1 hsdR17 r m phoA supE44 thi 1 gyrA9 6 relA1 tonA confers resistance to phage T1 Mach1 T1 Use this strain for general cloning and blue white screening without IPTG Strain is resistant to T1 bacteriophage F 480 lacZ AM15 AlacX74 hsdR r my ArecA1398 endA1 tonA confers r
14. SuperMix High Fidelity 100 reactions 10790 020 The PCR Optimizer Kit 100 reactions K1220 01 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 col One Shot MAX Efficiency DH5a T1 20 reactions 12297 016 Chemically Competent F col One Shot TOP10F Chemically 20 reactions C3030 03 Competent col 40 reactions C3030 06 Ampicillin 200 mg 11593 027 Kanamycin 5g 11815 024 25g 11815 032 100 mL 10 mg mL 15160 054 X gal 100 mg 15520 034 1g 15520 018 IPTG 1g 15529 019 S 0 C Medium 10x 10 mL 15544 034 PureLink Quick Plasmid Miniprep Kit 50 reactions K2100 10 PureLink Quick Gel Extraction Kit 50 reactions K2100 12 Chemical safety WARNING 32 Appendix D Safety GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain SDSs see the Documentation and Support section in this document Minimize contact with che
15. all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following In the U S e U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety e Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html e Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials e Additional information about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO_CDS_CSR_LYO_2004_11 en 33 Obtaining supp
16. and conditions of all applicable Limited Use Label Licenses INFORMATION FOR EUROPEAN CUSTOMERS The Mach1 T1 coli strain is genetically modified to carry the acZAM15 AsdR 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 TRADEMARKS All trademarks are the property of Thermo Fisher Scientific and its subsidiaries Life Technologies is a Thermo Fisher Scientific brand 2014 Thermo Fisher Scientific Inc All rights reserved Contents About this UM a a aaa aaa a are a EAEAN AAA AANA AN ENANA ANNAN EA NEN ANAA A AANA ANAN aN 4 Prod ctinformMatiO ssi eens ccsnccecatan cous eten tres etancuusccdscvesetedennteteadaceettansacctatasceutad sa teasadasaucatanteacatendaas 5 Contents de a eee dee eee 5 Description of the Systematic debited 9 o tatateetas cunt etna teccutas cuted satectadasceucoumaienaentantl 10 Produce RIDROdU Cs reee fhe neos ctas artos ohn Mi e a ohne Acted Cotes Achat eth dcl ts os 10 Perform the TOPO Cloning reaction ccccccccsscscsesscsesecsesescsesessesesscssssssesassessssesesacsesacsesacaesecacsecacseacsesaees 11 Transform One Shot competent CelS ccccccsccsssscscseescsesecsesescsesescsessssesessssesassesscsesesaesesacsesecaesecsesecaeseacas 13 Transform One Shot Mach1 T1 competent C US c ccccccsscscsscscseecsesecsc
17. ay be suitable 1 After amplification with a proofreading polymerase place vials on ice and add 0 7 1 unit of Taq 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 the vials at 72 C for 8 10 minutes do not cycle 3 Place the vials 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 proofreading polymerase After purification add Taq polymerase buffer dATP and 0 5 unit of Tag polymerase Incubate the reaction for 10 15 minutes at 72 C and use in the TOPO Cloning reaction 27 Recipes LB Luria Bertani medium and plates 28 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 the solution to cool to 55 C and add antibiotic if needed 50 pg mL of either ampicillin or kanamycin 4 Store at room temperature
18. ay 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 HCl pH 8 Incomplete extension during PCR Be sure to include a final extension step of 7 30 minutes during PCR Longer PCR products will need a longer extension time Cloning large inserts greater than 1 kb Try one or all of the following e Increase amount of insert e Incubate the TOPO Cloning reaction longer e Gel purify the insert see page 25 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 7ag polymerase page 27 Use the Zero Blunt PCR Cloning Kit to clone blunt PCR products Cat no K2800 20 PCR cloning artifacts false positives TOPO Cloning is very efficient for small fragments less than 100 bp present in certain PCR reactions Gel purify your PCR product page 25 PCR product does not contain sufficient 3 A overhangs even though you used 7ag polymerase Increase the final extension time to ensure all 3 ends are adenylated Taq polymerase is less efficient at adding a nontemplate 3 A next to another A 7agis most efficient at adding a nontemplate 3 A next toaC You may have to redesign your primers so that they contain a 5 G
19. com Other kits for plasmid DNA purification are also suitable for use 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 29 pCR 2 1 TOPO vector or page 30 pCR I TOPO vector for sequence surrounding the TOPO TA Cloning site For the full sequence of either vector visit www lifetechnologies com support or contact Technical Support page 32 Continued on next page Analyze transformants continued Analyze transformants by PCR IMPORTANT Long term storage 20 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 e PCR SuperMix High Fidelity see page 31 e Appropriate forward and reverse PCR primers 20 uM each Procedu
20. creases 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 you must dilute the TOPO Cloning reaction before transforming electrocompetent cells see the following sections Read the following information carefully e 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 e For TOPO Cloning and transformation of electrocompetent E coli salt must also be included in the TOPO Cloning reaction but th
21. dred colonies Pick 10 white or light blue colonies for analysis see Analyze Positive clones page 19 Do not pick dark blue colonies Transform One Shot DH5a T1 TOP10 and TOP10F competent cells Introduction Required materials Prepare for transformation Protocols to transform One Shot DH5a T1 TOP10 and TOP10F competent E coli are provided in this section Both chemical transformation and electroporation protocols are provided If you are transforming Mach1 T1 cells refer to the section entitled Transform One Shot Mach1 T1 competent cells pages 14 15 If using other competent cells follow manufacturer s instructions Components required but not supplied e The TOPO Cloning reaction from Perform the TOPO Cloning Reaction step 2 on page 12 e LB plates containing 50 pg mL ampicillin or 50 pg mL kanamycin e 40mg mL X gaL in dimethylformamide DMF e 100 mM IPTG in water for use with TOP10F e 15 mL snap cap plastic culture tubes sterile electroporation only e 42 C water bath e 37 C shaking and non shaking incubator e General microbiological supplies e g plates spreaders Components supplied with the kit e 0 C medium For each transformation you will need one vial 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
22. e amount of salt must be reduced to 50 mM NaCl 2 5 mM MgCl in order to prevent arcing After performing the TOPO Cloning reaction and prior to electroporation dilute the reaction 4 fold to achieve the proper salt concentration Continued on next page Perform the TOPO Cloning reaction continued Set Up the TOPO Cloning reaction Perform the TOPO Cloning reaction Note The following table describes how to set up your TOPO Cloning reaction 6 uL for eventual transformation into either chemically competent or electrocompetent TOP10 or chemically competent DH50 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 21 Note The red color of the TOPO vector solution is normal and is used to visualize the solution Reagent Volume Fresh PCR product 0 5 4 uL Salt Solution 1 uL Water add to a total volume of 5 uL TOPO vector 1 uL Final Volume 6 uL Store all reagents at 20 C when finished Salt solutions and water can be stored at room temperature or 4 C 1 Mix the reaction gently and incubate for 5 minutes at room temperature 22 23 C Note For most applications 5 minutes will yield sufficient 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 sec
23. esistance to phage T1 TOP10 Use this strain for general cloning and blue white screening without IPTG F mcrA A mrr hsdRMS mcrBC 80lacZAM15 AlacX74 recAl araD139 A ara leu 7697 galU galK rpsL Str endA1 nupG TOP10F This strain over expresses the Lac repressor lacl1 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 recAl araD139 A ara leu 7697 galU galK rpsL Str endA1 nupG Information for non U S customers using Mach1 T1 cells 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 Description of the system TOPO TA Cloning How Topoisomerase works Experimental outline TOPO TA Cloning provides a highly efficient 5 minute one step cloning strategy TOPO Cloning for the direct insertion of Taq polymerase amplified PCR products into a plasmid vector No ligase post PCR procedures or PCR primers containing specific sequences are required The plasmid pCR II TOPO vector or pCR 2 1 TOPO vec
24. ies Diluting 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 50 80 pL for 0 1 cm cuvettes or 100 200 uL for 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 Analyze transformants Analyze positive clones Sequencing Take 2 6 white or light blue colonies and culture them overnight in LB medium containing 50 pg mL ampicillin or 50 pg 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 pre warmed LB medium containing 50 pg mL ampicillin or 50 pg mL kanamycin before isolating the plasmid For optimal results we recommend inoculating as much of a single colony as possible Isolate plasmid DNA using PureLink Quick Plasmid Miniprep Kit supplied with Cat nos K4500 02 and K4510 02 or available separately see page 3131 The plasmid isolation protocol is included in the manual supplied with the PureLink Quick Plasmid Miniprep Kit and is also available from www lifetechnologies
25. instead of a 5 T Brownstein et al 1996 Appendix A Support protocols Purify PCR products Introduction Smearing multiple banding primer dimer artifacts or large PCR products greater than 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 Two simple protocols are described in this section Using the The PureLink Quick Gel Extraction Kit page 31 allows you to rapidly purify PCR PureLink Quick products from regular agarose gels Gel Extraction Kit 1 Equilibrate a water bath or heat block to 50 C Excise 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 3 Weigh the gel slice 4 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 5 Incubate the tube at 50 C for 15 minutes Mix every 3 minutes to ensure gel dissolution After the gel slice appears dissolved incubate the
26. ion on a prewarmed selective plate To ensure even spreading of small volumes add 20 pL 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 Analyze Positive Clones on page 19 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 protocol ampicillin selection For more information on selecting a transformation protocol refer to page 13 Note Warm LB plates containing ampicillin to 37 C prior to spreading 1 Add 4 pL of the TOPO Cloning reaction from Perform the TOPO Cloning Reaction step 2 page 12 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 pg mL ampicillin and incubate overnight at 37 C An efficient TOPO Cloning reaction should produce several hun
27. logies com support 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 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 l 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 35 Headquarters a 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit lifetechnologies com support or email techsupportlalifetech com technologies lifetechnologies com 24 February 2014
28. micals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Handle chemical wastes in a fume hood Ensure use of primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage After emptying a waste container seal it with the cap provided Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Biological hazard safety WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow
29. o a final volume of 49 pL Tag Polymerase 1 unit pL 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 following Note products Note 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 25 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 see page 31 incorporates many of the recommendations found in this reference Perform the TOPO Cloning reaction Introduction Note Using salt solution in the TOPO Cloning reaction 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 vector 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 14 18 before beginning If this is the first time you have TOPO cloned perform the control reactions on pages 22 23 in parallel with your samples We have found that including salt 200 mM NaCl 10 mM MgCl in the TOPO Cloning reaction in
30. onds may be sufficient For large PCR products greater than 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 Select a One Shot chemical transformation protocol on page 13 Note You may store the TOPO Cloning reaction at 20 C overnight TOPO TA Cloning Kits are optimized to work with One Shot Competent E coli available from Life Technologies Use of other competent cells may require further optimization Performing the control TOPO Cloning reaction is recommended as this control when used with the supplied protocol will demonstrate high cloning efficiencies Additionally transforming a control plasmid is highly recommended to confirm transformation efficiencies when using alternative competent cells not supplied by Life Technologies Transform One Shot competent cells Introduction Select a One Shot chemical transformation protocol IMPORTANT Recommendation After performing the TOPO Cloning reaction transform your pCR 2 1 TOPO or pCR II TOPO construct into the competent E coli General guidelines for transformation are provided below For transformation into competent E coli supplied with your kit refer to Transform One Shot Mach1 T1 competent cells pages 14 15 or Transform One Shot DH5a T1 TOP10 and TOP10F competent cells pages 16 18 depending on the compe
31. 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 pg 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 Appendix B Vectors Map of pCR 2 1 TOPO pCR 2 1 TOPO The following map shows the features of the pCR 2 1 TOPO vector and the map sequence surrounding the TOPO Cloning site Restriction sites are labeled to indicate the actual cleavage site The arrow indicates the start of transcription for T7 polymerase The sequence of the pCR 2 1 TOPO vector is available from www lifetechnologies com support or by contacting Technical Support page 32 M13 Reverse aimee ATG find lll Kpn l Sac Banh Spe l CAG GAA ACA GCT ATG ACC ATG ATT ACG CCA AGC TTG GTA CCG AGC TCG GAT CCA CTA GTC CTT TGT CGA TAC TGG TAC TAA TGC GGT TCG AAC CAT GGC TCG AGC CTA GGT GAT Be l Eco l EGOR l GTA ACG GCC GCC AGT GTG CTG GAA TTC GCC CTIE Ye E ano GGC GAA TIC TGC CAT TGC CGG CGG TCA CAC GAC CTT AAG CGG GA TTC CCG CTT AAG ACG ECOR V Bsr l Nor l xpo l Neil Xba l Apa l AGA TAT CCA TCA CAC TGG CGG CCG CTC GAG CAT GCA TCT AGA GGG CCC AAT TCG CCC TAT TCT ATA GGT AGT GTG ACC GCC GGC GAG CTC GTA CGT AGA TCT CCC GGG TTA AGC GGG ATA 4 T7 Promoter M13 Forward 20 Primer AGT GAG TCG TAT TAC AAT
32. orm One Shot DH5a T1 TOP10 and TOP10F competent cells continued One Shot electroporation protocol Note Add 18 pL of water to 6 pL of the TOPO Cloning reaction from Perform the TOPO Cloning reaction step 2 on page 12 Mix gently Note The TOPO Cloning reaction must be diluted in this step to prevent arcing Transfer 2 uL of the diluted TOPO Cloning reaction from step 1 of this procedure into a vial of One Shot Electrocompetent E coli and mix gently Do not mix by pipetting up and down Carefully transfer the solution into a 0 1 cm cuvette avoid formation of bubbles Electroporate your samples using your own protocol and your electroporator Note If you have problems with arcing see the following Note Immediately add 250 uL of room temperature S O C medium Transfer the solution into 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 onto a pre warmed selective plate and incubate overnight at 37 C To ensure even spreading of small volumes add 20 pL 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 Analyze positive clones on page 19 Do not pick dark blue colon
33. ort Obtaining SDS Obtaining Certificates of Analysis Limited product warranty 34 Documentation and Support For the latest services and support information for all locations go to www lifetechnologies com support At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support techsupport lifetech com e Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents e Obtain information about customer training e Download software updates and patches Safety Data Sheets SDSs are available at www lifetechnologies com support The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www lifetechnologies com support and search for the Certificate of Analysis by product lot number which is printed on the box Life Technologies Corporation and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechno
34. r 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 pL 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 Analyze positive clones on page 19 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 see page 13 Note It is essential that LB plates containing ampicillin are pre warmed prior to spreading 1 Add 4 pL of the TOPO Cloning reaction from Perform the TOPO Cloning reaction step 2 on page 12 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 pre warmed 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 Analyze positive clones on page 19 Do not pick dark blue colonies Continued on next page Transf
35. re 1 For each 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 10 colonies and resuspend them individually in 50 uL of the PCR cocktail from step 1 of this procedure 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 30 cycles For the final extension incubate at 72 C for 10 minutes Store at 4 C Visualize by agarose gel electrophoresis If you have problems obtaining transformants or the correct insert perform the control reactions described on pages 22 23 to help troubleshoot your experiment After identifying the correct clone be sure to prepare 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 on LB plates containing 50 ug mL ampicillin or 50 pg 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 5 Store at 80 C Optimize the TOPO Cloning reaction Faster subcloning More transformants Clone dilute PCR products The high efficiency of TOPO Cloning technology allows you
36. sececsesecsesecsesesacsesecsesecsesecaeseeeeaes 14 Transform One Shot DH5a T1 TOP10 and TOP10F competent cells c ccccscecesessesseeecsesescteteeeeeees 16 A alyze tran OIO ATE Ne E nens deme a te Coenen ttn o tae tant Caen acta Laat canes AA ner a O 19 Optimize the TOPO Cloning reaction cccccccssscsssscsesecscsesscsesesscsesessesessesesassessssessssssesacsesacassacscsecacseacseseees 21 Perform the control reactions taste bid a a Sade ade oe 22 Appendix A Support protocol ccccceeeeeeeeee ee eeeeee eens eee eeaaaaa ee eeeee sees aaa a eeeeeeeeaaaaaaaaeeeeeeseeeeaaaaaes 25 PURI Fe CRD OCLC LS ees 0 e e e ce a a St er ee e er es i hk 25 Adding 3 A overhangs post amplification ooooconccinnnncccnoncnoncnoncnn nono nonn nono ncnn nono nnnnnnnnn non nro n ran rra nrnn rra ninos 27 O 28 Appendix B Ve CtOr cccceeeeeeeee ee eeeee eee e cece eae aaaa eee eee eeeeaa dee eeeeeeeeaaaaaaaeeeeeeeeeaaaaaaaaeeeeeseseeaaaaaaeeeeeees 29 ME GR 2 A A shoal ee wich chepMoar ets cede ct 29 Maipo pER MAD cds 30 Appendix C Ordering information ccccccccceeeeeeeee eee e cece eee eae eee eeeeeee aaa eeeeeeeeaaaaaaaaeeeeeesseeeaaaaaees 31 AD PONCE Diz Safety iinne cae ceacesieapacemveaunsericapivewsidteteoudecueteadisuctwicansctanedieqettuarsaeetivaermeeienet 32 ChemicalsatelYeo cs nieto oe da o a e en 32 Biological hazard SY a a Pah TAN al O a e Cet ad 33 Documentation And Syp OT e E a a a a aa aa T a aa a aaa a Eaa
37. sy A Invitrogen by L fe technologies TOPO TA Cloning Kit Five minute cloning of 7ag polymerase amplified PCR products Catalog numbers pCR 2 1 TOPO vector K4500 01 K4500 40 K4500 J10 K4510 20 K4520 01 K4520 40 K4550 01 K4550 40 K4560 01 K4560 40 K4500 02 K4510 22 450641 Catalog numbers pCR I TOPO vector K4600 01 K4600 J10 K4600 40 K4610 20 K4620 01 K4620 40 K4650 01 K4650 40 K4660 01 K4660 40 Document Part Number 250184 Publication Number MAN0000047 Revision A 0 Now with 25 more TOPO reactions For Research Use Only Not for use in diagnostic procedures technologies Information in this document is subject to change without notice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF IMPORTANT LICENSING INFORMATION These products may be covered by one or more Limited Use Label Licenses By use of these products you accept the terms
38. tent E coli you wish to transform To transform another competent strain refer to the manufacturer s instructions 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 protocol clone large PCR products greater than 1000 bp use kanamycin as the selective agent see the following IMPORTANT obtain transformants as quickly as rapid chemical transformation possible protocol 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 and transformation reactions resulting in transformants that are ampicillin resistant and white but are not the desired construct Transform One Shot Mach1 T1 competent cells Introduction Pro
39. to streamline the cloning process If you routinely clone PCR products and wish to speed up the process consider the following 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 After adding 2 pL 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 Incubate the salt supplemented TOPO Cloning reaction for 20 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 Increase the amount of the PCR product Incubate the TOPO Cloning reaction for 20 30 minutes Concentrate the PCR product 21 Perform the control reactions Introduction Before starting Produce the control PCR product 2
40. tocols to transform One Shot Mach1 T1 chemically competent E coli are provided in this section If you are transforming cells other than Mach1 T1 cells refer to the section entitled Transform One Shot DH5a T1 TOP10 and TOP10F competent cells pages 16 18 If using other competent cells follow manufacturer s instructions Note 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 T1 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 ampicillin or kanamycin selective media Required materials Components required but not supplied e The TOPO Cloning reaction from Perform the TOPO Cloning reaction step 2 on page 12 e LB plates containing 50 pg 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 e General microbiological supplies e g plates spreaders Components supplied with the kit e S O C medium Prepare for For each transformation you will need one vial of competent cells and two transformation selective plates e Equilibrate a water bath to 42 C e Warm the vial of S 0 C medium from
41. tor 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 Taq 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 on CCCTT NAGGG GGGAR PCR Product Sree P Topoisomerase e Produce your PCR product e Set up the TOPO cloning reaction mix together the PCR Product and TOPO vector e Incubate for 5 minutes at room temperature e Transform the TOPO cloning reaction into One Shot Competent Cells or equivalent e Select and analyze 10 white or light blue colonies for insert Methods
42. uct Use only chemically competent cells for transformation 1 Electrophorese all of your PCR reaction on a low melt TAE agarose gel 0 8 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 12 Incubate the TOPO Cloning reaction at 37 C for 5 10 minutes This is to keep the agarose melted Transform 2 4 uL directly into competent One Shot cells using one of the methods described on pages 13 18 Cloning efficiency may decrease with purification of the PCR product You may wish to optimize your PCR to produce a single band Adding 3 A overhangs post amplification Introduction Required materials Procedure Note 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 This section describes a simple method to clone these blunt ended fragments e Taq polymerase e A heat block equilibrated to 72 C e Phenol chloroform optional e 3Msodium acetate optional e 100 ethanol optional e 80 ethanol optional e TE buffer optional This is just one method for adding 3 adenines Other protocols m
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