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Fast-FusionTM Cloning Kit
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1. 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 Accessories VII Limited Use License and Warranty l Introduction The GeneCopoeia Fast Fusion Cloning Kit provides a rapid method for cloning your PCR products 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 are no restriction sites 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 fideli
2. 1M NaCl 1ml 2M glucose filter sterilized 1ml 2M Mg stock filter sterilized Add Bacto tryptone Bacto yeast extract NaCl and KCI to 97ml of distilled water Stir to dissolve Autoclave and cool to room temperature Add 2M Mg2 stock and 2M glucose each to a final concentration of 20mM Bring the volume to 100ml with sterile distilled water The final pH should be 7 0 2M Mg stock 20 33 g MgCl2 6H20 24 65 g MgSQq 7H20 LB medium per liter 10g Bacto tryptone 5g Bacto yeast extract 5g NaCl Adjust the pH to 7 5 with NaOH Autoclave to sterilize For LB plates include 15g agar prior to autoclaving Accessorial products Description Catalog STK200 10 STK200 20 GCl 5a Chemically Competent E coli Cells 10 tubes 20 tubes STK300 10 STK300 20 GCI L3 Chemically Competent E coli Cells 10 tubes 20 tubes Fast Fusion Cloning Kit User Manual VIII 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 resol
3. C overnight Pick colonies for analysis Fast Fusion Cloning Kit User Manual 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 working properly before you Call us for help 1 Problem Few or no colonies obtained from transformation Possibility Solution Check the control reaction There should be at least 100 colonies from Competent cells efficiency is insufficient l ee competent cells with efficiencies greater than 10 cfu ug DNA solution impurity Purify the DNA by gel purification etc Check with known concentration DNA standards concentrate the DNA Low DNA concentration in reaction to greater than 20 ng uL Check your primers to ensure the products provide corresponding bases Primer sequences are incorrect of homology Homologies longer than 20 bp give the best results Don t use less than Not enough homology l Da l 12 bp if your competent cell efficiency is below 10 cfu ug Incomplete 3 ends generated by PCR Increase the elongation time after the last PCR cycle Make sure the especially for proofreading polymerases dNTPs in the PCR reaction are not exhausted after PCR cycles Make sure DNA solution buffer contains no more than 0 2mM EDTA EDTA repression which will repress the assembly reaction Dilute TE buffer to 0 1x before us
4. d 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 recombinant 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
5. e length of the gene specific sequence Colonies formed 3000 Colonies formed 0 10 20 30 40 50 60 homologies a ee 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 3 i Forward Primer 5 NNNNNNNNNNNNNNN gt _ _ _ cx4 12345 678 9 101112131415 Linearized Vectors 5 NNNNNNNNNNNNNN 3 5 NNNNNNNNNNNNNNNNNN 3 with 5 Overhangs 3 NNNNNNNNNNNNNNNNNN S 3 NNNNNNNNNNNNNN 5 NNNNNNNNNNNNNNN 5 Reverse Primer Forward Primer 5 NNNNNNNNNNNNNNN gt n 1234567 89 101112131415 Linearized Vectors 5 NNNNNNNNNNNNNNNNNN 3 5 NNNNNNNNNNNNNNNNNN 3 with Blunt ends 3 NNNNNNNNNNNNNNNNNN 5S 3 NNNNNNNNNNNNNNNNNN 5 1514131211098 7654321 L _ J lt lt NNNNNNNNNNNNNNN 5 Reverse Primer Forward Primer 5 NNNNNNNNNNNNNNN gt s i 12345 67 8 9101112131415 Linearized Vectors 5 NNNNNNNNNNNNNNNNNNNNNN 3 5 NNNNNNNNNNNNNNNNNN 3 with 3 Overhangs 3 NNNNNNNNNNNNNNNNNN S 3 NNNNNNNNNNNNNNNNNNNNNN 5 1814131211109 87 65432 1 ee o NNNNNNNNNNNNNNN 5 Reverse Primer Fig 3 Primer with 15 bp homology in different vector ends Forwa
6. 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 Suite 101 Rockville MD 20850 1 301 762 0888 1 866 360 9531 inqui enecopoeia 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 9
7. ing it as DNA solution buffer Increase the incubation time to 30 min for homologies longer than 30 bp Too much homology 60 minutes is recommended for homologies longer than 50 bp 2 Problem There are many colonies after transformation but none of the plasmids contain inserts Possibility Solution Incomplete linearization of vector Digest vector completely generate incompatible overhangs gel purify your digestion product transform a no insert control to verify few background colonies can grow Large amounts of DNA more than 400 ng in the reaction will either slow down the reaction or compete with your assembled molecules in Too much DNA transformed transformation Scale to no more than 200 ng per 100 uL chemically competent cells Contamination of PCR template carrying 1 10 ng of plasmid template is usually sufficient for PCR reaction Digest the same antibiotic resistance the plasmid template with Dpn I or gel purify the PCR product Insert Vector ratio is too low Re determine the molar ratio add into the reaction as recommended 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 VI Accessories Composition of Buffers and Solutions TE Buffer 10 mM Tris Cl pH8 0 1 mM EDTA pH8 0 SOC medium 100ml 2 0g Bacto tryptone 0 5g Bacto yeast extract 1ml 1M EDTA pH8 0 0 25 ml 1M KCl 1ml
8. minate reaction Keep on ice until transformation l Transform 1 5 uL reaction product to competent cells l Pick colonies for screening ll Contents and Storage Contents and storage recommendations for the GeneCopoeia Fast Fusion Cloning Kit Cat Nos FFPC C020 and 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 20 C TE Buffer Dry ice or ice pack Stable for at least 12 months 1 x 10 uL 20 C Linearized pUC19 50 ng uL Dry ice or ice pack 3 x 10 uL Stable for at least 12 months 1 x 10 uL 20 C Positive Insert 100 ng L Dry ice or ice pack 3 x 10 uL 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 inse
9. ore 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 Note To obtain better precipitation efficiency for DNA molecules shorter than 200 bp incubate at 4 C for at least 30 minutes before centrifugation Re suspend the DNA by adding 10 20 uL 0 1xTE buffer diluted with ddH20 Cloning Reaction and Transformation Procedure Cloning Reaction Set up the following 10 uL cloning reaction on ice When using the GeneCopoeia Fast Fusion Cloning Kit for the first time GeneCopoeia strongly recommends including positive and negative control reactions in parallel with your cloning reactions The linearized pUC19 vector and positive insert provided in the kit have already been purified so there is no treatment needed before use Reagents Cloning reaction Negative control Positive control Linearized Vector 20 100 ng 1 uL 1 uL linearized pUC19 Target Insert 20 150 ng a 1 uL positive insert 10 x Clonase Buffer 1 uL 1 uL 1 uL Fast Fusion Clonase 1 uL 1 uL 1 uL ddH20 Add ddH20 to 10 uL 7 uL Note The recommended molar ratio of insert to vector should be 2 5 1 Use the table below as a guide Vector Insert ax 30 50 ng 200 2000 bp 20 100 ng For multi insert assembly reduce
10. rd Primer 5 CG ACT CTA GAG GAT CXX XXX XXX 3 GCC TGC AGG TCG ACT CTA GAG G ATC CCC GGG TAC CGA GCT CGA ATT 3 3 CGG ACG TCC AGC TGA GAT CIC CTA G GGG CCC ATG GOT GGA GOT TAA pUC19 Sequence 3 XXX XXX XXC TAG GGG CCC ATG GC 3 Reverse Primer Fig 4 Example of primer designed for the GeneCopoeia Fast Fusion system Primer sequences are shown in bold Underlined bases are homologous to the end of pUC19 vector digested by restriction enzyme BamH I X bases corresponding to the gene or sequence of interest 3 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 5 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 5 PCR inserts for Fast Fusion cloning Lane 1 Insert PCR purified by QP reagent Lane 2 Insert PCR without purification Lane3 4 Nonspecific amplification in PCR reaction 1 2 3 4 1 1 Fast Fusion Cloning Kit User Manual 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 bef
11. rts 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 Check your primers following the guidelines below Each Fast Fusion primer consists of two parts 1 A sequence at the 5 end that is homologous to one end of the target vector or another insert and 2 a gene specific sequence at the 3 end that will specifically amplify the target insert Fig 3 4 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 Avoid complementarity within each primer to prevent hairpin structures and between primer pairs to avoid primer dimers The melting temperature Tm should be calculated based on the 3 end gene specific sequence of the primer not the entire primer GeneCopoeia recommends setting the Tm value of the primer between 55 C 65 C by adjusting th
12. scale of each insert The total scale of vector and inserts should keep around 200ng 10uL as optimal _ aa A O w yx 2 Fast Fusion Cloning Kit User Manual Homogenize the reaction mix by tapping the tube Centrifuge briefly to collect the liquid at the bottom of the tube Incubate at 25 C for 15 minutes Place the tube on ice until transformation Store the product directly below 20 C do not affect the success rate Optional Add 40 uL TE Buffer to terminate the reaction for long term storage 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 108 cfu ug 1 Transfer 1 2 uL of reaction mixture 5 10 uL after dilution with TE Buffer to 100 uL chemically competent cells Tap the tube gently for 2 3 times to mix them well Incubate on ice for 30 minutes Note 1 uL is usually sufficient for single insert cloning Increase volume for multi insert assembly Heat shock the cells for exactly 30 seconds at 42 C without shaking then immediately place the tubes on ice for 2 minutes Add 400 uL of room temperature S O C medium to the cells Cap the tubes and incubate at 37 C for 1 hour with or without shaking Spread 50 to 500 uL cells from each tube on pre warmed LB plates containing the appropriate antibiotics Incubate plates at 37
13. ty DNA polymerase Amplify insert with Generate your vector homology to vector SSS eee Enzyme digest o or PCR PCR and purify J Mm 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 l Fast Fusion Cloning Kit User Manual Working 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 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 or recombination sites 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 Seamless construction Final constructs have no extra base pairs remaining Protocol overview PCR and vector preparation PCR product purification Add Fast Fusion system incubate at 25 C for 15 min l Optional Add 4 volumes of TE buffer to ter
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