Fast-Fusion Cloning Kit
Contents
1. the tube 3 Spin down briefly to collect the reagents at the bottom of the tube Incubate at 25 C for 15 minutes 4 Add 40 uL TE Buffer to terminate the reaction or place the tube on ice until transformation 2 Transformation Transform competent E coli cells with your Fast Fusion products using the provided protocol below or by following the manufacturer s instructions GeneCopoeia recommends using high efficiency competent cells gt 10 cfu ug 1 Transfer 1 to 5 uL of reaction mixture 5 25 uL after dilution with TE Buffer to 100 uL chemically competent cells Incubate on ice for 30 minutes Note 1 uL is usually sufficient for single insert cloning Increase volume for multi insert assembly 2 Heat shock the cells for exactly 30 seconds at 42 C without shaking then immediately place the tubes on ice for 2 minutes 3 Add 400 uL of room temperature S O C medium to the cells 5 Fast Fusion Cloning Kit User Manual 4 Cap the tubes and incubate at 37 C for 1 hour with or without shaking 5 Spread 50 to 500 uL cells from each tube on pre warmed LB plates containing the appropriate antibiotics 6 Incubate plates at 37 C overnight 7 Pick colonies for analysis V Troubleshooting The tables below address two main problems encountered during Fast Fusion cloning along with their possible causes and suggested solutions Please perform the control reactions to confirm that the kit is working2. Expressway to Discovery Fast Fusion Cloning Kit For rapid and effective cloning of PCR products Cat No FFPC C020 20 reactions Cat No FFPC C060 60 reactions User Manual GeneCopoeia Inc 9620 Medical Center Drive Suite 101 Rockville MD 20850 USA 301 762 0888 866 360 9531 inquiry genecopoeia com www genecopoeia com 2014 GeneCopoeia Inc Fast Fusion Cloning Kit User Manual User Manual Fast Fusion Cloning Kit l Introduction ll Contents and Storage lll Key Steps IV Cloning Reaction and Transformation Procedure V Troubleshooting VI Limited Use License and Warranty I Introduction Fast Fusion Cloning Kit provides a rapid method for cloning your PCR product In just 15 minutes at room temperature any PCR fragment can be cloned into your linearized vector at will After a simple clean up step a PCR generated DNA fragment or other purified DNA fragment can be joined to a vector with overlapping ends Fig 1 Up to eight DNA fragments can be joined together in a single reaction Well prepared vectors generate almost 100 positive clones There is no restriction site required at the junction site Therefore your fragment of interest can be inserted at any position in the vector The linearized vector can be generated by either PCR or restriction enzyme digestion The PCR products can be produced by either Taq DNA polymerase or other high fidelity DNA polymerase Amplify insert wi
3. NNNNNNNN 3 5 NNNNNNNNNNNNNNNNNN 3 with 5 Overhangs 3 NNNNNNNNNNNNNNNNNN S _ 3 NNNNNNNNNNNNNN 5 NNNNNNNNNNNNNNN S Reverse Primer Forward Primer 5 NNNNNNNNNNNNNNN gt r Y 12345 67 89 101112131415 Linearized Vectors 5 NNNNNNNNNNNNNNNNNN 3 5 NNNNNNNNNNNNNNNNNN 3 with Blunt ends 3 NNNNNNNNNNNNNNNNNN 5 3 NNNNNNNNNNNNNNNNNN 5 1814131211109 87654321 ee lt NNNNNNNNNNNNNNN S Reverse Primer Forward Primer 5 NNNNNNNNNNNNNNN gt _ _ ci 123 45 67 8 9101112131415 Linearized Vectors 5 WNNNNNNNNNNNNNNNNNNNNNN 3 5 NNNNNNNNNNNNNNNNNN 3 with 3 Overhangs 3 NNNNNNNNNNNNNNNNNN 5 3 NNNNNNNNNNNNNNNNNNNNNN 5 1514131211109 8 7654321 US lt NNNNNNNNNNNNNNN 5 Reverse Primer Fig 3 Primer with 15 bp homology in different vector ends PCR amplification and purification Taq and other high fidelity DNA polymerases are all suitable for generating DNA fragments for Fast Fusion cloning After PCR analyze PCR products by electrophoresis on an agarose EtBr gel The QP reagent can be used when only a single band is present Fig 4 Gel purification is strongly recommended when nonspecific amplification is evident Quantify the purified fragments by measuring against a known DNA standard running in parallel Fig 4 PCR inserts for Fast Fusion cloning Lane 1 Insert PCR purified by QP reagent Lane 2 Insert PCR without purification L
4. ane3 4 Nonspecific amplification in PCR reaction Use of QP reagent The QP reagent can precipitate double stranded DNA longer than 100 bp excluding dNTPs primers and most of the polymerase Invert the QP reagent tube several times before use For 50 uL of PCR product add TE buffer to 100 uL followed by addition of 50 uL QP reagent Mix thoroughly by vortexing for 5 seconds Centrifuge the mixture at 15 000xg for 15 minutes and discard the supernatant Re centrifuge the tube for 10 seconds and remove all the remaining liquid at the bottom Fast Fusion Cloning Kit User Manual Note To obtain better precipitation efficiency for DNA molecules shorter than 200 bp incubate at 4 C for at least 30 minutes before centrifugation 4 Re suspend the DNA by adding 10 20 uL diluted TE Buffer Dilute TE Buffer to 10 with ddH20 IV Cloning Reaction and Transformation Procedure 1 Cloning Reaction 1 Set up the following cloning reaction on ice Component Volume Amount 10 x Clonase Buffer 1 uL Linearized vector 20 100 ng Insert 20 200 ng ddH2O0 Add ddH20 to 9 pL Note The recommended molar ratio of insert to vector should be 2 5 1 Use the table below as a guide vector insert Length Quantity Length Quantity 3k bp 20 50 ng 200 2000 bp 100 ng 5k bp 40 80 ng 2k 5k bp 100 150 ng 9k bp 50 100 ng gt 5k bp 150 200 2 Add 1 pL Fast Fusion clonase to the reaction and mix by tapping
5. ast Fusion system incubate at 25 C for 15 min l Add 5 volumes of TE buffer to terminate reaction keep on ice until transformation l Transform 1 5 uL reaction product to competent cells l Pick colonies for screening FFPC C060 are provided in the following table Contents Quantity Shipping temperature Storage temperature 1 x 20 uL 20 C Fast Fusion Clonase Dry ice or ice pack 3 x 20 uL Stable for at least 12 months 1 x 20 uL 20 C 10 x Clonase Buffer Dry ice or ice pack 3 x 20 uL Stable for at least 12 months 1 x 500 uL 20 C QP Reagent Dry ice or ice pack 3 x 500 uL Stable for at least 12 months 1 x 500 uL 20 C TE Buffer Dry ice or ice pack 3 x 500 uL Stable for at least 12 months Linearized pUC19 1x 10 uL 20 C Dry ice or ice pack 50 ng uL 3 x 10uL Stable for at least 12 months Positive Insert 1x10puL 20 C Dry ice or ice pack 100 ng L 3 x 10uL Stable for at least 12 months Fast Fusion Cloning Kit User Manual Additional materials required but not provided Clonable plasmid vector Taq or other high fidelity DNA polymerases DNA quantitation standard Restriction enzymes Gel purification kit Competent cells for transformation S 0 C medium LB plates with antibiotics Key Steps Vector preparation A well prepared vector can reduce your screening time Single enzyme digested vectors will self ligate resulting in a high background of plasmids lacking
6. inserts following transformation The best way to avoid this is to digest with two restriction enzymes followed by gel purification of the vector backbone For PCR generated vectors we recommend digestion with Dpn which will destroy plasmids that have been Dam methylated by replication in E coli Transform 50 100 uL of competent cells with 5 10 ng linearized vector as a negative control to determine the transformation background Primer design Primer design is critical for successful Fast Fusion cloning Homology must present at the ends you want to fuse e g vector and insert or multiple inserts For homologies less than 15 bp the transformation efficiency will vary depending on DNA structure Fig 2 GeneCopoeia strongly recommends including more than 15 bp of homology at each end for best results Check your homology following the guidelines below Fig 3 Colonies formed 6000 5000 4000 Colonies formed 3000 2000 1000 0 10 20 30 40 50 60 homologies bp Fig 2 Homologies affect cloning efficiency The number of colonies formed is calculated from 5 ng of pUC19 vector transformed after standard Fast Fusion reactions with inserts of indicated homologies Competent cells efficiency 2x 10 cfu ug Fast Fusion Cloning Kit User Manual Homologous Sequence Specific Sequence Forward Primer 5 NNNNNNNNNNNNNNN gt i i 12345678 9101112131415 Linearized Vectors 5 NNNNNN
7. ite 101 Rockville MD 20850 1 301 762 0888 1 866 360 9531 inquiry genecopoeia com GeneCopoeia Products are for Research Use Only Copyright 2014 GeneCopoeia Inc Trademarks GeneCopoeia OmicsLink Secrete Pair GLuc ON miTarget Fast Fusion GeneCopoeia Inc FFPC 140210 7
8. properly before you Call us for help 1 Problem Few or no colonies obtained from transformation Possibility Solution Competent cells efficiency is insufficient Check with control reaction There should be at least 100 colonies for competent cells over 10 cfu ug DNA solution impurity Purify the DNA by gel purification etc Low DNA concentration in reaction Check with known concentration DNA standards concentrate the DNA to over 20 ng L Primer sequences are incorrect Check your primers to ensure the products provide corresponding bases of homology with their neighbors Not enough homology Homologies over 20 bp give best results Don t use less than 12 bp if your competent cell efficiency is below 10 cfu ug Incomplete 3 ends generated by PCR especially for proofreading polymerases Increase the elongation time after the last PCR cycle Make sure the dNTPs in the PCR reaction are not exhausted after PCR cycles Too much homology Increase the incubation time to 30 min for homologies over 30 bp 60 minutes is recommended for homologies greater than 50 bp Too much DNA transformed High concentration of DNA over 400 ng in the reaction will either slow down the reaction or compete with your assembled molecules in transformation Scale to no more than 200 ng per 100 pL chemically competent cells 2 Problem There are many colonies after transforma
9. t DNA and genetic research Use of any part of the Product constitutes acceptance of the above terms Limited Warranty GeneCopoeia warrants that the Product meets the specifications described in the accompanying Product Datasheet If it is proven to the satisfaction of GeneCopoeia that the Product fails to meet these specifications GeneCopoeia will replace the Product In the event a replacement cannot be provided GeneCopoeia will provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to GeneCopoeia within 30 days of receipt of the Product GeneCopoeia s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price GeneCopoeia s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty GeneCopoeia does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose GeneCopoeia is committed to providing our customers with high quality products If you should have any questions or concerns about any GeneCopoeia products please contact us at 301 762 0888 20014 GeneCopoeia Inc GeneCopoeia Inc 9620 Medical Center Drive Su
10. th Generate your vector homology to vector Le Enzyme digest E or PCR PCR and purify EE MMM Mix with proper ratio Add Fast Fusion clonase Incubate at 25 C for 15 min Transform E coli Fig 1 Experimental workflow of single fragment insertion into a vector using the GeneCopoeia Fast Fusion Cloning Kit Work principle The GeneCopoeia Fast Fusion Cloning Kit inserts the fragment into the vector using two simultaneous steps a homology recognition b strand exchange and redundant strand degradation The gaps remaining in the recombinant Fast Fusion Cloning Kit User Manual strands will be repaired by E coli after transformation Key Advantages Fast and simple 1 minute for operation and 15 minutes for incubation at room temperature High efficiency Greater than 90 of colonies after transformation contain the correct insert s High adaptability No restriction site or recombination site needed insert fragments generated by either PCR or restriction enzyme digestion can be used Flexibility Multiple inserts can be assembled in one reaction Suitable for multi site mutagenesis e Seamless construction Final constructs have no extra base pairs remaining Protocol overview ll Contents and Storage Contents and storage recommendations for the GeneCopoeia Fast Fusion Cloning Kit Cat Nos FFPC C020 and PCR and vector preparation PCR product purification Add F
11. tion but none of the plasmids contain inserts Possibility Incomplete linearization of vector Solution Digest vector completely generate incompatible overhangs gel purify your digestion product transform a no insert control to verify few background colonies can grow Contamination of PCR template carrying the same antibiotic resistance 1 10 ng of plasmid template is usually sufficient for PCR reaction Digest the plasmid template with Dpn l or gel purify the PCR product Antibiotics expired or incorrect Do an empty incubation in 37 C to make sure the antibiotics are not expired Fast Fusion Cloning Kit User Manual Vil Limited Use License and Warranty Limited Use License Following terms and conditions apply to use of Fast Fusion Cloning Kit the Product If the terms and conditions are not acceptable the Product in its entirety must be returned to GeneCopoeia within 5 calendar days A limited End User license is granted to the purchaser of the Product The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product must not be resold repackaged or modified for resale or used to manufacture commercial products without prior written consent from GeneCopoeia This Product should be used in accordance with the NIH guidelines developed for recombinan
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