In-Fusion Dry-Down PCR Cloning Kit User Manual
Contents
1. 16 X References 17 Appendix A Control Vector Map and In Fusion Cloning Site 18 List of Figures Figure 1 The In Fusion Cloning Method 3 Figure 2 Flow Chart of the In Fusion Dry Down PCR Cloning Kit Protocols 4 Figure 3 Universal Primer Design for the In Fusion System 9 Figure 4 Examples of primers designed for In Fusion cloning 10 Figure 5 pUC19 Linearized Vector Map amp In Fusion Cloning Site 18 List of Tables Table I Recommended Nanograms of Vector per In Fusion Reaction 12 Table II Recommended Microliters of Cloning Enhancer Treated Insert per In Fusion Reaction 12 Table III Recommended In Fusion Reactions for Gel amp Spin Column Purified Inserts 13 Ta2. 8 C PCR Amplification of Insert 10 D Control Reaction 11 V In Fusion Procedure for Cloning Enhancer Treated Inserts 11 A Procedure for Treating Unpurified Single Band PCR Inserts with Cloning Enhancer 11 B In Fusion Cloning Procedure for Cloning Enhancer Treated Inserts 12 VI In Fusion Procedure for Gel amp Spin Column Purified PCR Inserts 13 A Procedure for Spin Column Purification of Multiple and Single Band PCR Inserts 13 B In Fusion Cloning Procedure for Gel amp Spin Column Purified PCR Inserts 13 VII Transformation Procedure 15 VIII Expected Results 16 IX Troubleshooting Guide
3. with a single specific band no background If you obtain PCR product with nonspecific background isolate the target fragment by gel extraction OR Spin Column purify Screen clones x x Recombinant vector M 1 2 M 1 2 Spin Column Protocol Treat with DpnI Figure 2 Flow Chart of the In Fusion Dry Down PCR Cloning Kit Protocols Clontech Laboratories Inc www clontech com Protocol No PT3941 1 A Takara Bio Company Version No PR9Z3434 5 In Fusion Dry Down PCR Cloning Kit User Manual I Introduction and Protocol Overview continued The In Fusion Enzyme promotes single strand annealing reactions SSA and in this manner can assemble DNA molecules that share short sequence overlaps or homologies at their ends such as a PCR amplified insert and a linearized vector When the In Fusion Enzyme is incubated with double stranded inserts and a linearized vector in the provided buffer the enzyme s exonuclease activity excises nucleotides from the 3 ends of the molecules exposing the overlapping sequence The exposed overlapping ends are free to anneal SSA forming non covalently joined molecules that undergo final repair within the target E coli strain The resulting product is an assembled vector and insert The reaction is seamless adding no more bases than you chose to have present If you generate nonspecific PCR products that contain additional unwanted background bands it is still possible to clon
4. at 4 C LB antibiotic plates Prepare LB medium as above but add 15 g L of agar before autoclaving Autoclave on liquid cycle for 20 min at 15 lb in2 Let cool to 55 C add antibiotic e g 100 g ml of ampicillin and pour into 10 cm plates After the plates harden then invert and store at 4 C SOC medium 2 Tryptone 0 5 Yeast Extract 10 mM NaCl 2 5 mM KCl 10 mM MgCl2 6H2O 20 mM glucose For 1 liter dissolve 20 g of tryptone 5 g of yeast extract and 0 5 g of NaCl in 950 ml of deionized H 1 2O Prepare a 250 mM KCl solution by dissolving 1 86 g of KCl in deionized H 2 2O for a total volume of 100 ml Add 10 ml of this stock KCl solution to the solution prepared in Step 1 Adjust pH to 7 0 with 5 M NaOH then bring the volume to 980 ml with deionized H 3 2O Prepare a 1 M solution of MgCl 4 2 by dissolving 20 33 g of MgCl2 6H2O in deionized H2O for a total volume of 100 ml Autoclave both solutions on liquid cycle at 15 lbs in 5 2 for 20 min Meanwhile make a 2 M solution of glucose by dissolving 36 g of glucose in deionized H 6 2O for a total volume of 100 ml Filter sterilize this solution Let the autoclaved solutions cool to about 55 C then add 10 ml of the filter sterilized 2 M glucose solution and 10 ml of 7 1 M MgCl2 Store at room temperature or 4 C NucleoSpin Extract II Kit Cat Nos 740609 50 amp 740609 250 PCR products do not need to be purified for successful
5. of background due to differences in cutting efficiency Generally speaking two enzymes cut better than any single enzyme Efficiency of digestion will always be better if the restriction enzyme sites are as far apart as possible In ad dition increasing the enzyme digestion time and digestion reaction volume will reduce the background Prepare a linearized vector as follows 1 We recommend cutting the vector with two different enzymes to reduce background unless there is only one site available for cloning 2 g Vector 10 l 10X Enzyme buffer 10 20 U Restriction enzyme X l Deionized water to 100 l 100 l Total Volume We recommend adding half the units of enzyme 2 5 5 U g at the beginning of the reaction Add the remaining enzyme units approximately 30 min later 2 Incubate your restriction digest as directed by the restriction enzyme supplier For many enzymes incubation from 3 hours to overnight can increase linearization and reduce background 3 After digestion purify the linearized vector using any available PCR purification kit We recommend using the NucleoSpin Extract II Kit Cat Nos 740609 50 amp 740609 250 4 Control Check the background of your vector by transforming 5 10 ng of the linearized and purified vector into Fusion Blue Competent Cells See Transformation Procedure Section VII If the background is high continue digesting the vector for a longer time after the additio
6. the vector and insert to the dry down reaction tube and incubate for 30 minutes For added convenience the dry down kits are available in multiple sizes For high throughput cloning a 96 well format is available Always store any unused dry down reaction tubes in a desiccator at 20 22 C Cloning Enhancer is included with catalog numbers 639607 639608 amp 639609 and is also sold separately in 25 50 amp 100 rxn sizes Cat Nos 639613 639614 amp 639615 respectively Some of our In Fusion Dry Down PCR Cloning Kits include Fusion Blue Competent Cells Cat Nos 639602 639604 amp 639609 We also offer cell free kits for users who wish to supply their own competent cells The In Fusion Dry Down PCR Cloning Kits are available in 8 reaction 24 reaction amp 96 reaction sizes with or without Cloning Enhancer or Com petent Cells Please see Section II for a list of components in each of our In Fusion Dry Down Kits Note Cutting wells from the 96 well plate may disturb the seals on remaining wells thereby damaging the efficacy of the dry down pellets If you do not plan on using all 96 pellets in the 96 rxn kit at one time we recommend that you instead purchase one of the 8 rxn or 24 rxn kits Attention In Fusion Dry Down PCR Cloning Kit User Manual Protocol No PT3941 1 www clontech com Clontech Laboratories Inc Version No PR9Z3434 A Takara Bio Company 6 Always store any unused In Fusion Dry Down Mix in a
7. 3 x strip of 8 96 pellets 96 well plate pUC19 Control Vector linearized 50 ng l 5 l 5 l 5 l 2 kb Control Insert 40 ng l 10 l 10 l 10 l Cloning Enhancer 50 l tube 50 l 1 tube 50 l 1 tube 200 l 4 tubes Fusion Blue Competent Cells 50 l tube 500 l 10 tubes Not Included Not Included SOC Medium 2 ml tube 4 ml 2 tubes Not Included Not Included Optically Clear PCR Cap Strips 1 3 12 Microseal A Film Not Included Not Included 1 Clontech Laboratories Inc www clontech com Protocol No PT3941 1 A Takara Bio Company Version No PR9Z3434 7 In Fusion Dry Down PCR Cloning Kit User Manual III Additional Materials Required The following materials are required but not supplied TE Buffer pH 8 0 required for diluting In Fusion reaction prior to transformation 10 mM Tris HCl 1 mM EDTA Sodium Acetate 3 M required only if concentrating DNA by precipitation Glycogen 20 g l required only if concentrating DNA by precipitation Ampicillin 100 mg ml stock required for the plating of the In Fusion control reaction LB Luria Bertani medium pH 7 0 for 1 liter 1 0 Bacto tryptone 10 g 0 5 Yeast extract 5 g 1 0 NaCl 10 g Dissolve ingredients in 950 ml of deionized H2O Adjust the pH to 7 0 with 5 M NaOH and bring the volume up to 1 L Auto clave on liquid cycle for 20 min at 15 lb in2 Store at room temperature or
8. I Wrong molar ratio The 2 1 molar ratio of PCR fragment to linear vector used in the In Fusion protocol may not have been optimal We recommend using between 100 ng and 300 ng of vector for cloning gel purified inserts Clontech provides an online tool to assist in determin ing the correct amount of insert and vector to achieve a 2 1 ratio http bioinfo clontech com infusion Primer sequences are incorrect Check primer sequences to ensure that they provide 15 bases of homology with the region flanking the insertion site see Section IV If you do not obtain the expected results use the following guide to troubleshoot your experiment To confirm that your kit is working properly perform the control reactions VIII Expected Results The positive control plates typically develop several hundred white colonies when using cells with a minimum competency of 1 x 108 cfu g The negative control plates should have few colonies The number of colonies on your experimental plates will depend on the amount and purity of the PCR product and linearized vector used for the In Fusion cloning reaction The presence of a low number of colonies on both plates typically a few dozen colonies is indicative of either transformation with too much of the reaction poor DNA quality or poor primer quality The presence of many hundreds of colonies on the negative control is indicative of contamination with a PCR template plasmid carr
9. In Fusion cloning If however multiple nonspecific bands are observed See Section IV C we recommend that you first gel purify your fragment of interest using the NucleoSpin Extract II Kit Section VI B This kit can also be used to purify your linearized vector Cloning Enhancer Cat Nos 639613 639614 amp 639615 Optional Competent Cells Some of the In Fusion Dry Down PCR Cloning Kits include Fusion Blue Competent Cells If your In Fusion Dry Down Kit does not include competent cells we strongly recommend the use of competent cells with a transformation efficiency gt 1 x 108 cfu g Clontech sells Fusion Blue chemically Competent Cells separately in 24 transformation Cat No 636700 and 96 transfor mation Cat No 636758 formats Clontech also offers Stellar Electrocompetent Cells Cat No 636765 for cloning Both Fusion Blue and Stellar cell lines offer a transformation efficiency gt 1 x108 cfu g In Fusion Dry Down PCR Cloning Kit User Manual Protocol No PT3941 1 www clontech com Clontech Laboratories Inc Version No PR9Z3434 A Takara Bio Company 8 IV PCR and Experimental Preparation PLEASE READ ENTIRE PROTOCOL BEFORE STARTING A Preparation of Linearized Vector by Restriction Digestion To achieve a successful In Fusion reaction you must first generate a very pure linearized vector with very low background of uncut vector present Restriction enzymes will generate different amounts
10. In Fusion reaction Adjust the amount of your input DNA if the size of your vector or PCR fragment is differ ent from above Clontech provides an online tool to assist in determining the correct amount of insert and vector to achieve a 2 1 ratio http bioinfo clontech com infusion 1 Mix your purified PCR insert and vector together at a 2 1 molar ratio in 10 l of deionized H2O If necessary co precipitate your DNA as follows Mix the vector and PCR insert together at the correct molar ratio in a 50 100 l volume Add 1 l glycogen 20 g l and 1 10 volume sodium acetate 3 M and mix Then add 3 volumes of ethanol 20 C to precipitate and then centrifuge at maximum speed for 10 min at 4 C Wash the pellet once with 70 ethanol Air dry the pellet and suspend the DNA pellet in H2O to a volume of 10 l Protocol Note Gel purified inserts do not require DpnI treatment prior to spin column purification Attention IMPORTANT DO NOT treat gel purified or spin column purified inserts with the Cloning Enhancer Figure 2 TABLE III RECOMMENDED IN FUSION REACTIONS FOR GEL amp SPIN COLUMN PURIFIED INSERTS Rxn Component Cloning Rxn Negative Control Rxn Positive Control Rxn PCR insert 50 200 ng 50 ng Linearized vector 100 300 ng 100 ng 100 ng Deionized water to 10 l to 10 l to 10 l Use 1 l of the 50 ng l solution of linearized pUC19 Control Vector included in the kit Use 2
11. In FusionTM Dry Down PCR Cloning Kit User Manual Cat Nos 639602 639604 639605 639606 639607 639608 amp 639609 PT3941 1 PR9Z3434 Published January 2010 United States Canada 800 662 2566 Asia Paci c 1 650 919 7300 Europe 33 0 1 3904 6880 Japan 81 0 77 543 6116 Clontech Laboratories Inc A Takara Bio Company 1290 Terra Bella Ave Mountain View CA 94043 Technical Support US E mail tech clontech com www clontech com User Manual In Fusion Dry Down PCR Cloning Kit User Manual Protocol No PT3941 1 www clontech com Clontech Laboratories Inc Version No PR9Z3434 A Takara Bio Company 2 Table of Contents I Introduction and Protocol Overview 3 II List of Components 6 III Additional Materials Required 7 IV PCR and Experimental Preparation 8 A Preparation of Linearized Vector by Restriction Digestion 8 B PCR Primer Design
12. TC GAC GGT AC C GGA CAT ATG CCC GGG AAT T 3 A B C Clontech Laboratories Inc www clontech com Protocol No PT3941 1 A Takara Bio Company Version No PR9Z3434 11 In Fusion Dry Down PCR Cloning Kit User Manual A Procedure for Treating Unpurified Single Band PCR Inserts with Cloning Enhancer Before setting up the In Fusion cloning reaction treat unpurified PCR products e g inserts containing a single band of desired size without nonspecific background as follows Add 2 l of Cloning Enhancer to 5 l of the PCR reaction 1 Incubate at 37 C for 15 minutes then at 80 C for 15 minutes in a PCR thermal cycler If you used more 2 than 100 ng of DNA as a template in the PCR reaction extend the 37 C incubation step to 20 minutes If you are using water baths or heat blocks instead of a thermal cycler preset them at 37 C and 80 C respectively and extend each of the incubation steps to 20 25 minutes 3 Proceed with the In Fusion Cloning Procedure for Cloning Enhancer Treated PCR Inserts Section V B If you cannot proceed immediately store treated PCR reactions at 20 C until you are ready IMPORTANT DO NOT treat purified PCR products with the Cloning Enhancer BREAK V In Fusion Procedure for Cloning Enhancer Treated Inserts D Control Reactions When using the In Fusion kit for the first time we strongly recommend that you perform the positive and negative control reactions in parallel with yo
13. al Protocol No PT3941 1 www clontech com Clontech Laboratories Inc Version No PR9Z3434 A Takara Bio Company 10 IV PCR and Experimental Preparation continued C PCR Amplification of Insert It is important to use only 10 100 ng of plasmid DNA as a PCR template However if you are amplifying a pool of cDNA the amount of template DNA depends on the relative abundance of the target message in your mRNA population For best results we recommend using our Advantage HD Polymerase Mix Cat No 639241 which offers high fidelity efficient amplification of long gene segments gt 1 kb and an automatic hot start that reduces nonspecific products When PCR cycling is complete analyze your PCR product by electrophoresis on an agarose EtBr gel to con firm that you have obtained a single DNA fragment and to estimate the concentration of your PCR product Quantify the amount of DNA by measuring against a known standard or molecular weight marker ladder run on the same gel The linearized vector provided in the kit is useful for this purpose Protocol IMPORTANT If you generate a single clean PCR product with little background you can purify your PCR amplified insert using spin columns see Section VI or simply treat your insert with Cloning Enhancer Cat Nos 639613 639614 amp 639615 see Section V If multiple bands are observed we recommend that you first gel purify your fragment see Section VI We reco
14. ble IV Troubleshooting Guide for In Fusion Experiments 16 Contact Us For Assistance Customer Service Ordering Technical Support Telephone 800 662 2566 toll free Telephone 800 662 2566 toll free Fax 800 424 1350 toll free Fax 650 424 1064 Web www clontech com Web www clontech com E mail orders clontech com E mail tech clontech com Clontech Laboratories Inc www clontech com Protocol No PT3941 1 A Takara Bio Company Version No PR9Z3434 3 In Fusion Dry Down PCR Cloning Kit User Manual The In Fusion Dry Down PCR Cloning Kits are designed to join multiple pieces of DNA which have 15 base pairs of homology at their linear ends A typical use for this technology would be cloning of PCR products into vectors without the need for restriction enzyme cleavage of the vector or insert and without the use of ligase or blunt end polishing Using our proprietary In Fusion Enzyme this kit rapidly generates precise constructs with inserts in the desired orientation Furthermore the In Fusion Dry Down PCR Cloning Kits w Cloning Enhancer allow direct use of an unpurified PCR product in the cloning reaction In Fusion is high throughput compatible and universal it works with any insert and any vector at any restriction site and allows you to disregard restriction sites within the insert The linearized vector can be generated using
15. clontech com Protocol No PT3941 1 A Takara Bio Company Version No PR9Z3434 13 In Fusion Dry Down PCR Cloning Kit User Manual A Procedure for Spin Column Purification of Multiple and Single Band PCR Inserts Depending on whether your PCR reaction contains a single band of desired size or yields multiple bands or nonspecific background on a gel prepare your PCR product as follows see Figure 2 If nonspecific background or multiple bands are visible on your gel isolate your target fragment by 1 gel extraction then spin column purify see Step 2 If you obtain a single band of the desired size add 1 l of DpnI to 50 l of the PCR reaction and incubate at 37 C for 60 min Spin column purify your PCR product e g insert by using a silica based purification system such 2 as NucleoSpin Extract II Kit Cat Nos 740609 50 amp 740609 250 During purification avoid nuclease contamination and exposure of the DNA to UV light for long periods of time 3 After spin column purification following either DpnI treatment or gel extraction proceed with the In Fusion Cloning Procedure for Spin Column Purified PCR Inserts Section VI B B In Fusion Cloning Procedure for Gel amp Spin Column Purified PCR Inserts In general maximum cloning efficiency is achieved when using a 2 1 molar ratio of insert vector Typically 100 ng of a 4 to 5 kb linearized vector plus 50 ng of a 1 kb PCR fragment is found to work well in a 10 l
16. d a known amount of your linearized vector Competent cells should give gt 1 x 108 cfu g See Section VIII for Expected Results 1 Transform competent cells with 2 5 l of diluted reaction mixture as follows a Using Fusion Blue Competent Cells Thaw one vial of frozen Fusion Blue Competent Cells on ice Tap tube gently to ensure that the cells are suspended Add 2 5 l of the diluted reaction mixture to the cells Mix gently to ensure even distribution of the DNA solution Leave the tube on ice for 30 min Heat shock the cells in a water bath at 42 C for 45 sec and then place them directly on ice for 1 min After a heat shock add 450 l of SOC medium to the cells and then incubate at 37 C for 60 min while shaking at 250 rpm b If using other competent cells with In Fusion Kits follow the transformation protocol provided with the cells DO NOT add more than 5 l of diluted reaction to 50 l of competent cells and proceed to Step 3 2 Take 1 10 of the cells 25 50 l from each transformation bring the volume to 100 l with SOC medium and then spread on separate LB plates containing 100 g ml of ampicillin or other appropriate antibiotic for your cloning vector 3 Centrifuge the remaining mix at 6000 rpm for 5 min resuspend the cells in 100 l fresh SOC and spread the remainder of the transformation mix on a separate LB plate containing the appropriate antibiotic Incubate all plates a
17. d into each primer start at the 5 end of each DNA strand in the linearized vector The region of homology for a particular primer consists of bases that are complementary to the rst 15 bases at the 5 end of a particular DNA strand This means that the bases complementary to 5 overhangs are included in the primer sequence but the bases in 3 overhangs are not 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 NNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNN NNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN Figure 3 Universal Primer Design for the In Fusion System Successful insertion of a PCR fragment requires that the PCR insert share 15 bases of homology with the ends of the linearized vector This sequence homology is added to the insert through the PCR primers For vectors with sticky ends bases complementary to 5 overhangs are included in the primer sequence bases in the 3 overhangs are not See Figure 4 for specific examples An online tool is also provided to assist in primer design and can be found at www clontech com ifprimers In Fusion Dry Down PCR Cloning Kit User Manu
18. desiccator at 20 22 C Store Fusion Blue Competent Cells at 70 C Store all other components at 20 C II List of Components The amount of vector provided in the In Fusion Dry Down Kits is sufficient for performing only the control reactions Transformation efficiency gt 1 0 x 108 cfu g Competent cells are only provided with Cat Nos 639602 639604 amp 639609 In Fusion Dry Down PCR Cloning Kits Components Cat Nos 639602 639604 639606 649605 Rxns 8 rxns 24 rxns 24 rxns 96 rxns In Fusion Dry Down Mix Component Amounts 8 pellets 1 x strip of 8 24 pellets 3 x strip of 8 24 pellets 3 x strip of 8 96 pellets 96 well plate pUC19 Control Vector linearized 50 ng l 5 l 5 l 5 l 5 l 2 kb Control Insert 40 ng l 10 l 10 l 10 l 10 l Test Plasmid 0 2 ng l 2 ng 4 ng Not Included Not Included Fusion Blue Competent Cells 50 l tube 500 l 10 tubes 1250 l 25 tubes Not Included Not Included SOC Medium 2 ml tube 4 ml 2 tubes 12 ml 6 tubes Not Included Not Included Optically Clear PCR Cap Strips 1 3 3 12 Microseal A Film Not Included Not Included Not Included 1 In Fusion Dry Down PCR Cloning Kits w Cloning Enhancer Components Cat Nos 639609 639607 639608 Rxns 8 rxns 24 rxns 96 rxns In Fusion Dry Down Mix Component Amounts 8 pellets 1 x strip of 8 24 pellets
19. e immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research No right under any other patent claim such as method claims in U S Patents Nos 5 210 015 5 487 972 5 994 056 and 6 171 785 and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is hereby conveyed by the purchase of this prod uct expressly by implication or by estoppel This product is for research use only Diagnostic uses require a separate license from Roche Further information on purchasing licenses may be obtained by contacting the Director of Licens ing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA U S Patent No 5 436 149 for LA Technology is owned by Takara Bio Inc TaqStart Antibody and other Hot Start Antibodies are licensed under U S Patent No 5 338 671 DH5 DH5a DH10B and ElectroMax are trademarks and Max Efficiency is a registered trademark of Invitrogen Corporation Microseal is a registered trademark of Bio Rad Laboratories Inc NucleoSpin and NucleoTrap are registered trademarks of Macherey Nagel GmbH and Co KG Clontech the Clontech Logo and all other trademarks are the property of Clontech Laboratories Inc unless noted oth erwise Clontech is a Takara Bio Company 2010 Clontech Laboratorie
20. e using the In Fusion method Rather than using the Cloning Enhancer nonspecific PCR inserts should be gel purified and then used directly in an In Fusion cloning reaction When gel purifying an insert DO NOT use the Cloning Enhancer treatment Figure 2 illustrates the differences in the experimental workflow required for cloning pure clean PCR inserts versus PCR inserts containing nonspecific products You can choose to spin column purify PCR products if you would rather not use the Cloning Enhancer The In Fusion method does not require the presence of nor is it affected by A overhangs so you can use any thermostable polymerase for amplification including proofreading enzymes To obtain high yields of small fragments lt 4 kb we recommend using the Advantage HF 2 enzyme provided in Clontech s Advantage HF 2 PCR Kits Cat Nos 639123 amp 639124 For accurate efficient production of long PCR inserts gt 1 kb we recommend using our Advantage HD Polymerase Mix Cat No 639241 The In Fusion Dry Down PCR Cloning Kit Formats The In Fusion Dry Down PCR Cloning Kits provide reaction components in a lyophilized format for maximal convenience and flexibility All of the necessary cloning reaction materials except the vector and the PCR insert are supplied in the reaction tube thus reducing the variability between reactions Simply pre treat your PCR insert with the Cloning Enhancer or gel purify add 10 l of distilled water containing
21. f the remaining tubes b Add 10 l of vector insert DNA H2O from Step 3 to each In Fusion Dry Down pellet Mix well by pipetting up and down 5 Incubate the reaction for 15 min at 37 C followed by 15 min at 50 C then place on ice 6 Dilute the In Fusion reaction mixture with 40 l TE buffer pH 8 and mix well 7 Proceed with Transformation Section VII If you cannot transform cells immediately store cloning reactions at 20 C until you are ready BREAK TABLE I RECOMMENDED NANOGRAMS OF VECTOR PER IN FUSION REACTION Vector Size Nanograms Recommended lt 4 kb 100 ng 4 to 6 kb 100 to 150 ng 6 to 10 kb 200 ng gt 10 kb Up to 400 ng Protocol TABLE II RECOMMENDED MICROLITERS OF CLONING ENHANCER TREATED INSERT PER IN FUSION REACTION Insert Length Microliters of Cloning Enhancer Treated Insert lt 1 kb 1 l 1 to 4 kb 1 to 2 l 4 to 8 kb 4 l 8 to 12 kb 7 l If you have a very weak PCR product we recommended adding more of the Cloning Enhancer treated insert up to 7 l Note Improved efficiency is observed for In Fusion reactions with a total volume of vector insert that is 5 l or less Attention Attention IMPORTANT Before proceeding to the cloning reaction be sure your target insert has been pretreated with the Cloning Enhancer as described in Section V A DO NOT follow this procedure if your insert has been purified Clontech Laboratories Inc www
22. ing Attention In Fusion Dry Down PCR Cloning Kit User Manual Protocol No PT3941 1 www clontech com Clontech Laboratories Inc Version No PR9Z3434 A Takara Bio Company 16 IX Troubleshooting Guide TABLE IV TROUBLESHOOTING GUIDE FOR IN FUSION EXPERIMENTS A No or Few Colonies Obtained from Transformation Description of Problem Explanation Solution Low transformation efficiency Transformed with too much In Fusion reaction mixture Do not add more than 5 l of diluted In Fusion reaction or 1 l of undiluted In Fusion reaction to 50 l of competent cells See Sec tion VII for details Suboptimal PCR product Repeat PCR amplification and purify product using a different method of purification Alternatively perform phenol chloroform extraction on your original PCR product followed by ethanol precipitation Bacteria were not com petent Check transformation efficiency You should obtain gt 1 x 108 cfu g otherwise use fresh competent cells Low quality DNA fragments Low DNA concentration in reaction It is imperative to obtain the highest DNA concentration possible in your In Fusion reaction Either the amount of vector or the amount of PCR fragment was too low For Cloning Enhancer treated inserts we recommend using between 100 ng and 400 ng of vector depending on its size see Table I In the case of gel purified inserts we recom mend using between 100 and 300 ng of vector Table II
23. l of the 40 ng l solution of 2 kb Control Insert included in the kit Protocol VI In Fusion Procedure for Gel amp Spin Column Purified PCR Inserts In Fusion Dry Down PCR Cloning Kit User Manual Protocol No PT3941 1 www clontech com Clontech Laboratories Inc Version No PR9Z3434 A Takara Bio Company 14 VI In Fusion Procedure for Gel amp Spin Column Purified PCR Inserts continued BREAK 2 Set up the In Fusion reactions a Peel back the aluminum seal s from the tube s you plan on using and take care to avoid disturbing the seals of the remaining tubes b Add 10 l of vector insert DNA H2O from Step 1 to each In Fusion Dry Down pellet Mix well by pipetting up and down 3 Incubate the reaction for 15 min at 37 C followed by 15 min at 50 C then place the tube on ice 4 Dilute the In Fusion reaction mixture with 40 l TE buffer pH 8 and mix well 5 Proceed with Transformation Section VII If you cannot transform cells immediately store cloning reactions at 20 C until you are ready Clontech Laboratories Inc www clontech com Protocol No PT3941 1 A Takara Bio Company Version No PR9Z3434 15 In Fusion Dry Down PCR Cloning Kit User Manual VII Transformation Procedure In addition to the cloning reaction we recommend that you perform positive and negative control trans formations which consist of a transformation control using a circular vector of known concentration an
24. mmend NucleoSpin Extract II Cat Nos 740609 50 amp 740609 250 for gel purification of PCR ampli fied inserts Attention Figure 4 Examples of primers designed for In Fusion cloning The above figure shows examples of primers de signed with recognition sites for restriction enzymes that generate 5 overhangs Panel A blunt ends Panel B and 3 overhangs Panel C The primer sequences are shown in bold The Xs represent bases correspond ing to the gene or sequence of interest Additional nucleotides indicated with a black box have been added to each primer in order to reconstruct the restriction sites They are not part of the 15 bases of sequence homology HindIII 5 ATA CAT TAT ACG AAG TTA TCA G AGC TTT CTA GAC CAT TCG TTT GGC G 3 SalI 3 TAT GTA ATA TGC TTC AAT AGT CAG CT AA GAT CTG GTA AGC AAA CCG C 5 5 G AAG TTA TCA GTC GAC XXX XX 3 3 X XXX XXT TCG AAA GAT CTG GTA 5 5 Forward Primer 3 Reverse Primer vector sequence SmaI 5 TCA GTC GAC GGT ACC GGA CAT ATG CCC GGG AAT TCC TGC AGG ATC CGC T 3 SmaI 3 AGT CAG CTG CCA TGG CCT GTA TAC GGG CCC TTA AGG ACG TCC TAG GCG A 5 5 ACC GGA CAT ATG CCC GGG XXX 3 3 XXX GGG CCC TTA AGG ACG TCC 5 5 AG TTA TCA GTC GAC GGT ACC XXX 3 KpnI KpnI 3 GTA ATA TGC TTC AAT AGT CAG CTG C CA TGG CCT GTA TAC GGG CCC TTA A 5 3 XXX CCA TGG CCT GTA TAC GGG CC 5 5 CAT TAT ACG AAG TTA TCA G
25. n be used to transform E coli PCR product Amplify your gene of interest Gene speci c primers with 15 bp extensions homologous to vector ends The In Fusion Enzyme creates single stranded regions at the ends of the vector and PCR product which are then fused due to the 15 bp homology Any Linearized vector Recombinant vector Single tube protocol In Fusion Dry Down PCR Cloning Kit User Manual Protocol No PT3941 1 www clontech com Clontech Laboratories Inc Version No PR9Z3434 A Takara Bio Company 4 I Introduction and Protocol Overview continued Add the following to each In Fusion Dry Down pellet x l Vector x l Insert x l dH2O 10 l Total Volume Set up the In Fusion cloning reaction Cloning Enhancer Protocol Amplify your gene of interest Determine the volume of Cloning Enhancer treated insert and linearized vector to use in the In Fusion reaction Add 2 l of Cloning Enhancer to 5 l of PCR product and incubate 15 min at 37 C 15 min at 80 C Incubate cloning reaction 15 min at 37 C 15 min at 50 C 15 bp 15 bp PCR product Mix the purified PCR insert and vector together at a 2 1 molar ratio Transform competent E coli with the diluted reaction mixture Dilute reaction with TE Buffer Generate a linearized vector Design gene specific primers with 15 bp extensions homologous to vector ends Treat with Cloning Enhancer If you obtain pure PCR product
26. n of more restriction enzyme s Incubate 2 hours to overnight Gel purify the remainder of the vector and transform again B PCR Primer Design Primer design and quality are critical for the success of the In Fusion reaction You can join two or more fragments e g vector and insert or multiple inserts as long as they share 15 bases of homology at each end Figure 3 outlines the guidelines for primer design Figure 4 gives specific examples of primers Therefore design PCR primers that will generate the homologous region in the PCR product during the amplification Every In Fusion primer must serve two purposes it should contain an In Fusion Ready homologous sequence and be gene specific The 15 base pairs towards the 5 end of the primer must match the 15 base pairs at the linear end of the DNA fragment to which it will be joined The 3 end of the primer is the gene specific portion of the primer The 3 end of the primer must have a melting temperature Tm suitable for PCR If you are using software to design your primers please note that the Tm should be calculated based upon the 3 gene specific end of the primer and NOT the entire primer If the calculated Tm is too low increase the length of the gene specific portion of the primer until you reach a Tm of between 58 65 C The Tm difference between the forward and reverse primers should be 4 C or you will not get good amplification Protocol Note To en
27. oduct is not a single distinct band then it may be necessary to gel purify the PCR product to ensure cloning of the correct insert See Section VI for more information X References D Arpa P 2003 Strategies for Cloning PCR Products In PCR Primer A Laboratory Manual Eds Dieffen bach C W amp Dveksler G S Cold Spring Harbor Laboratory Press NY pp 405 420 Fisher C L amp Pei G K 1997 Modification of a PCR based site directed mutagenesis method BioTech niques 23 4 570 574 Innis M A Gelfand D H Sninsky J J amp White T J Eds 1990 PCR Protocols A Guide to Methods and Applications Academic Press Inc San Diego CA Sambrook J amp Russell D W 2001 Molecular Cloning A Laboratory Manual Cold Spring Harbor Labo ratory Press NY Weiner M P Costa G L Shoettlin W Cline J Mathur E amp Bauer J C 1994 Site directed mutagen esis of double stranded DNA by the polymerase chain reaction Gene 151 119 123 In Fusion Dry Down PCR Cloning Kit User Manual Protocol No PT3941 1 www clontech com Clontech Laboratories Inc Version No PR9Z3434 A Takara Bio Company 18 Appendix A Control Vector Map and In Fusion Cloning Site pUC19 2690 bp lacZ part B pUC ori Ampr In Fusion Cloning Site lacZ part A MCS GAATTCGAGC TCGGTACCCG GGGATC CTTAAGCTCG AGCCATGGGC CCCTAG SmaI XbaI XmaI AccI HincII SacI SalI SphI Pa
28. restriction enzymes single or double cut or by PCR The In Fusion PCR Cloning Method The In Fusion method is simple and efficient First design PCR primers that have at least 15 bases of ho mology with sequences flanking the desired site of insertion in the cloning vector refer to Section IV B of this manual Using those primers amplify the DNA insert by PCR Directly treat the PCR product with our proprietary Cloning Enhancer Cat Nos 639613 639614 amp 639615 or spin column purify Then combine the PCR product with the linearized vector in the In Fusion cloning reaction In general the In Fusion reaction consists of a simple 30 min incubation of the PCR product with the lin earized cloning vector followed by transformation into E coli Figure 1 Each reaction generates inserts in the correct orientation and precise constructs without incorporation of any additional nucleotides This procedure can be easily automated With many vectors optional blue white selection on X Gal plates can be used to screen out rare non linearized vector background The protocol works best using high quality highly concentrated PCR generated DNA fragments with minimal background I Introduction and Protocol Overview Figure 1 The In Fusion Cloning Method During the 30 min incubation the In Fusion Enzyme creates single stranded regions at the ends of the vector and PCR product which are then fused due to the 15 bp homology The resulting clone ca
29. rtial BamHI Site EcoRI HindIII PstI KpnI CTAGGAGA TCTCAGCTGG ACGTCCGTAC GTTCGAACC GATCCTCT AGAGTCGACC TGCAGGCATG CAAGCTTGG In Fusion Cloning Site Partial BamHI Site Figure 5 pUC19 Linearized Vector Map amp In Fusion Cloning Site Sequence and digest information is available in PT4065 5 and can be downloaded from our website at www clontech com manuals Clontech Laboratories Inc www clontech com Protocol No PT3941 1 A Takara Bio Company Version No PR9Z3434 19 In Fusion Dry Down PCR Cloning Kit User Manual Notice to Purchaser Clontech products are to be used for research purposes only They may not be used for any other purpose including but not limited to use in drugs in vitro diagnostic purposes therapeutics or in humans Clontech products may not be transferred to third parties resold modified for resale or used to manufacture commercial products or to provide a service to third parties without written approval of Clontech Laboratories Inc This product is covered by U S Patent No 7 575 860 and European Patent No EP1741787 Fusion Blue Cells are manufactured and tested by Novagen for Clontech Laboratories Inc Novagen is a registered trademark of EMD Biosciences Inc LIMITED LICENSE Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US 5 079 352 and 6 127 155 The purchase of this product includes a limited non transferabl
30. s Inc
31. sure that background is low in a critical cloning experiment first gel purify the vector Protocol Clontech Laboratories Inc www clontech com Protocol No PT3941 1 A Takara Bio Company Version No PR9Z3434 9 In Fusion Dry Down PCR Cloning Kit User Manual IV PCR and Experimental Preparation continued Clontech provides an online tool at www clontech com ifprimers that simplifies the In Fusion PCR primer design for standard cloning reactions Simply provide your vector sequence the restriction enzyme s used to linearize the vector if that is the chosen method for linearization and the primer sequence required to amplify your region of interest We generally use desalted oligos in PCR reactions However oligo quality can depend on the vendor and varies from lot to lot If your oligo supply is particularly poor i e has many premature termination prod ucts or your PCR primer is longer than 45 nucleotides you may need to use PAGE purified oligos but in general we find that this is unnecessary Brackets indicate bases to be included in the 15 b region of homology Linearized Vectors with 5 Overhangs Linearized Vectors with Blunt ends Linearized Vectors with 3 Overhangs Forward Primer Reverse Primer append with your speci c sequence Reverse Primer Forward Primer Forward Primer Reverse Primer Guidelines for universal primer design To determine the 15 b homology sequence to be incorporate
32. t 37 C overnight 4 The next day pick individual isolated colonies from each experimental plate Isolate plasmid DNA using a standard method of your choice e g miniprep To determine the presence of insert analyze DNA by restriction digest or PCR screening Protocol Note If your In Fusion Dry Down Kit does not include competent cells we strongly recommend the use of competent cells with a transformation efficiency gt 1 x 108 cfu g Clontech sells Fusion Blue Competent Cells separately in 24 transformation Cat No 636700 and 96 transformation Cat No 636758 formats Clontech also offers Stellar Electrocompetent Cells Cat No 636765 for cloning Both Fusion Blue and Stellar cell lines offer a transformation efficiency gt 1 x 108 cfu g Note DO NOT add more than 5 l of diluted reaction to 50 l of competent cells More is not better Using too much of the reaction mixture inhibits the transformation For example 0 5 1 l of an undiluted In Fusion reaction in 50 l of cells typically yields over 1 000 colonies while 2 l of the same reaction will yield fewer than 100 colonies Since it can be difficult to pipette 1 l accurately e g if you are using yellow tips with a p20 pipettor we have suggested Section V C 6 and Section VI C 4 that you dilute the In Fusion reaction with TE buffer before perform ing the transformation especially if you wish to use a small volume of competent cells e g HTP clon
33. ur In Fusion cloning reaction Performing the control reactions will verify that the system is working properly The 2 kb Control Insert included in the In Fusion Dry Down PCR Cloning Kits has already been purified so there is no need for further treatment prior to the cloning reaction To perform the control reactions proceed with the In Fusion Cloning Procedure for Spin Column Purified PCR Inserts Section VI A Note The amount of control vector and control insert provided in the In Fusion Liquid Kits is sufficient for performing only the control reactions IV PCR and Experimental Preparation continued Protocol In Fusion Dry Down PCR Cloning Kit User Manual Protocol No PT3941 1 www clontech com Clontech Laboratories Inc Version No PR9Z3434 A Takara Bio Company 12 V In Fusion Procedure for Cloning Enhancer Treated Inserts continued B In Fusion Cloning Procedure for Cloning Enhancer Treated Inserts 1 Use Table I to determine the final amount of linearized vector to use in your In Fusion reaction 2 Use Table II to determine the final amount of Cloning Enhancer treated PCR insert from Section V A to use in your In Fusion reaction 3 Mix the insert and vector and adjust the final volume to 10 l using deionized H2O The final volume must not exceed 10 l 4 Set up the In Fusion reaction a Peel back the aluminum seal s from the tube s you plan on using and take care to avoid disturbing the seals o
34. ying antibiotic resistance Clontech Laboratories Inc www clontech com Protocol No PT3941 1 A Takara Bio Company Version No PR9Z3434 17 In Fusion Dry Down PCR Cloning Kit User Manual IX Troubleshooting Guide continued TABLE IV TROUBLESHOOTING GUIDE FOR IN FUSION EXPERIMENTS B Large Numbers of Colonies Contained No Insert Description of Problem Explanation Solution Large numbers of colo nies obtained with no insert Incomplete linearization of your vector It is important to remove any uncut vector prior to use in the In Fusion reaction If necessary recut your vector and gel purify Contamination of In Fusion reaction by plas mid with same antibiotic resistance If your insert was amplified from a plasmid closed circular DNA vector may have carried through purification and contaminated the cloning reaction To ensure the removal of any plasmid contamination we recommend linearizing the vector template before performing PCR Alternatively the PCR product can be treated with DpnI to remove the parental vector template after PCR amplification Weiner et al 1994 Fisher et al 1997 Plates too old or con tained incorrect antibiotic Be sure that your antibiotic plates are fresh lt 1 month old Check the antibiotic resistance of your fragment C Clones Contain Incorrect Insert Large number of colonies contain incorrect insert PCR product containing nonspecific sequences If your PCR pr
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