Gateway® Cloning ProtocolsThe Basics of Gateway® Reaction∙BP reaction—to create a Gateway® entry clone∙LR reaction—to create a Gateway® expression clone∙One tube format—to create a Gateway® expression clone from a PCR product∙Gateway® Vector Conversion—converting your favorite cloning vectors to Gateway® TechnologyTOPO® TA Cloning - To Create a Gateway® Entry CloneStep One - Produce PCR productProduce PCR products using Taq polymerase and your own protocol. Endthe PCR reaction with a final 7 to 30 minute extension step.Step Two - Perform the TOPO® Cloning Reaction1. Set up one of the following TOPO® Cloning reactions using thereagents in the order shown. For electroporation, dilute SaltSolution 4-fold to prepare Dilute Salt Solution.Reagent Chemical Txn ElectroporationFresh PCR product0.5 to 4 µl0.5 to 4 µlSalt Solution 1 µl--Dilute SaltSolution-- 1 µlSterile Water to a final volume of 5µlto a final volume of 5µlTOPO® Vector 1 µl 1 µlTotal volume6µl6µl2. Mix gently and incubate for 5 minutes at room temperature.3. Place on ice and proceed to transform One Shot® chemically competent E. coli, belowStep Three - Transform One Shot® Chemically Competent E. coli1. For each transformation, thaw one vial of One Shot® E. coli cells on ice.2. Add 2 µl of the TOPO® Cloning reaction into a vial of One Shot® chemically competent E. coli and mix gently.3. Incubate on ice for 5 to 30 minutes.4. Heat-shock the cells for 30 seconds at 42°C without shaking. Immediatelytransfer the tube to ice.5. Add 250 µl of room temperature S.O.C. Medium.6. Incubate at 37°C for 1 hour with shaking.7. Spread 10-50 µl of bacterial culture on a prewarmed LB agar plate containing100 µg/ml spectinomycin, and incubate overnight at 37°C.The Basics of Gateway® ReactionsBP ReactionCreating a Gateway® entry clone from an att B-flanked PCR product is an easy 1 hour reaction. See below for an overview of the set-up. For more detailed information, refer to the manual.1.Add the following components to a 1.5 ml tube at roomtemperature and mix:attB-PCR product (=10 ng/µl; final amount ~15-150 ng) 1-7 µlDonor vector (150 ng/µl) 1 µlTE buffer, pH 8.0 to 8 µl2.Thaw on ice the BP Clonase™ II enzyme mix for about 2 minutes.Vortex the BP Clonase™ II enzyme mix briefly twice (2 secondseach time).3.To each sample (Step 1, above), add 2 µl of BP Clonase™ IIenzyme mix to the reaction and mix well by vortexing brieflytwice. Microcentrifuge briefly.4.Return BP Clonase™ II enzyme mix to -20°C or -80°C storage.5.Incubate reactions at 25°C for 1 hour.6.Add 1 µl of the Proteinase K solution to each sample toterminate the reaction. Vortex briefly. Incubate samples at37°C for 10 minutes.Transformation1.Transform 1 µl of each BP reaction into 50 µl of One Shot ®OmniMAX ™ 2 T1 Phage-Resistant Cells (Catalog no. C8540-03).Incubate on ice for 30 minutes. Heat-shock cells by incubatingat 42°C for 30 seconds. Add 250 µl of S.O.C. Medium andincubate at 37°C for 1 hour with shaking. Plate 20 µl and 100µl of each transformation onto selective plates. Note: Anycompetent cells with a transformation efficiency of >1.0 × 108 transformants/µg may be used.2.Transform 1 µl of pUC19 DNA (10 ng/ml) into 50 µl of One Shot ®OmniMAX ™ 2 T1 Phage-Resistant Cells as described above. Plate20 µl and 100 µl on LB plates containing 100 µg/ml kanamycin,or the appropriate selection marker for your donor vector.Expected ResultsAn efficient BP recombination reaction will produce >1500 colonies if the entire BP reaction is transformed and plated.LR ReactionTransferring your gene from a Gateway® entry clone to destination vector is an easy 1 hour reaction. See below for an overview of the set-up. For more detailed information, refer to the manual.1.Add the following components to a 1.5 ml tube at roomtemperature and mix:Entry clone (50-150 ng) 1-7 µlDestination vector (150 ng/µl) 1 µlTE buffer, pH 8.0 to 8 µl2.Thaw on ice the LR Clonase ™ II enzyme mix for about 2 minutes.Vortex the LR Clonase ™ II enzyme mix briefly twice (2 secondseach time).3.To each sample (Step 1, above), add 2 µl of LR Clonase ™IIenzyme mix to the reaction and mix well by vortexing brieflytwice. Microcentrifuge briefly.4.Return LR Clonase ™ II enzyme mix to -20°C or -80°C storage.5.Incubate reactions at 25°C for 1 hour.6.Add 1 µl of the Proteinase K solution to each sample toterminate the reaction. Vortex briefly. Incubate samples at37°C for 10 minutes.TransformationFollow the protocol as indicated for the BP reaction, except use the appropriate selection marker for the LB plates suited to your destination vector (typically 100 µg/ml ampicillin).Expected ResultsAn efficient LR recombination reaction will produce >5000 colonies if the entire LR reaction is transformed and plated.One Tube FormatIf you want to transfer your attB-flanked PCR product directly into an expression clone, you can easily combine the BP and LR reactions using the following protocol. This will potentially eliminate the transformation and DNA isolation of the Gateway® entry clone.1.In a 1.5 ml microcentrifuge tube, prepare the following 15 µlBP reaction:attB DNA (50-100 ng) 1.0-5.0 µlattP DNA (pDONR™ vector, 150 ng/µl) 1.3 µlBP Clonase™ II enzyme mix 3.0 µlTE Buffer, pH 8.0 add to a final volume of 15 µl2.Mix well by vortexing briefly and incubate at 25°C for 4hours.Note: Depending on your needs, the length of the recombinationreaction can be extended up to 20 hours. An overnightincubation typically yields 5 times more colonies than a 1 hour incubation. Longer incubation times are recommended for largeplasmids (=10 kb) and PCR products (=5 kb).3.Remove 5 µl of the reaction to a separate tube and use thisaliquot to assess the efficiency of the BP reaction (seebelow).4.To the remaining 10 µl reaction, add:Destination vector (150 ng/µl) 2.0 µlLR Clonase™ II enzyme mix 3.0 µlFinal volume 15 µl5.Mix well by vortexing briefly and incubate at 25°C for 2hours.Note: Depending on your needs, the length of the recombinationreaction can be extended up to 18 hours.6.Add 2 µl of proteinase K solution. Incubate at 37°C for 10minutes.7.Transform 50 µl of the appropriate competent E. coli with 1 µlof the reaction.8.Plate on LB plates containing the appropriate antibiotic toselect for expression clones.Assessing the Efficiency of the BP Reaction1.To the 5µl aliquot obtained from “One-Tube” Protocol, Step 3,above, add 0.5 µl of proteinase K solution. Incubate at 37°Cfor 10 minutes.2.Transform 50 µl of the appropriate competent E. coli with 1 µlof the reaction. Plate on LB plates containing the appropriateantibiotic to select for entry clones.Gateway® Vector ConversionConverting your favorite set of cloning vectors to Gateway® Technology is a fairly straightforward protocol, and will ultimately allow you to streamline your cloning and expression process.To convert your cloning vector to a Gateway® destination vector, you will:1.Choose the appropriate reading frame cassette to use dependingon your needs.2.Linearize the vector you wish to convert with a restrictionenzyme of choice. If you use a restriction enzyme thatgenerates an overhang, you will need to blunt the ends.3.Remove the 5' phosphates from the vector using calf intestinalalkaline phosphatase.4.Ligate the reading frame cassette into your vector using T4 DNAligase.5.Transform the ligation reaction into One Shot® ccdB Survival™Competent E. coli and select for transformants.6.Analyze transformants.。