GatewayGateway CloningCloning ProtocolsProtocolsTheThe BasicsBasics ofof GatewayGateway ReactionReactionBP reactionto create a Gateway entry cloneLR reactionto create a Gateway expression cloneOne tube formatto create a Gateway expression clone from a PCR productGateway Vector Conversionconverting your favorite cloning vectors to Gateway TechnologyTOPOTOPO TATA CloningCloning - - ToTo CreateCreate a a GatewayGateway EntryEntry CloneCloneStepStep OneOne - - ProduceProduce PCRPCR productproduct Produce PCR products using Taq polymerase and your own protocol. End the PCR reaction with a final 7 to 30 minute extension step. StepStep TwoTwo - - PerformPerform thethe TOPOTOPO CloningCloning ReactionReaction 1. Set up one of the following TOPO Cloning reactions using the reagents in the order shown. For electroporation, dilute Salt Solution 4-fold to prepare Dilute Salt Solution.ReagentReagentChemicalChemical TxnTxnElectroporationElectroporation Fresh PCR product0.5 to 4 l0.5 to 4 l Salt Solution1 l- Dilute Salt Solution-1 lSterile Waterto a final volume of 5 lto a final volume of 5 l TOPO Vector1 l1 l Total volume6l6l2. Mix gently and incubate for 5 minutes at room temperature. 3. Place on ice and proceed to transform One Shot chemically competent E. coli, belowStepStep ThreeThree - - TransformTransform OneOne ShotShot ChemicallyChemically CompetentCompetent E.E. colicoli 1. For each transformation, thaw one vial of One Shot E. coli cells on ice. 2. Add 2 l 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 42C without shaking. Immediately transfer the tube to ice. 5. Add 250 l of room temperature S.O.C. Medium. 6. Incubate at 37C for 1 hour with shaking. 7. Spread 10-50 l of bacterial culture on a prewarmed LB agar plate containing 100 g/ml spectinomycin, and incubate overnight at 37C.TheThe BasicsBasics ofof GatewayGateway ReactionsReactionsBPBP ReactionReaction Creating a Gateway entry clone from an attB-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 room temperature and mix: attB-PCR product (=10 ng/l; final amount 15-150 ng) 1-7 l Donor vector (150 ng/l) 1 l TE buffer, pH 8.0 to 8 l 2. Thaw on ice the BP Clonase II enzyme mix for about 2 minutes. Vortex the BP Clonase II enzyme mix briefly twice (2 seconds each time). 3. To each sample (Step 1, above), add 2 l of BP Clonase II enzyme mix to the reaction and mix well by vortexing briefly twice. Microcentrifuge briefly. 4. Return BP Clonase II enzyme mix to -20C or -80C storage. 5. Incubate reactions at 25C for 1 hour. 6. Add 1 l of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly. Incubate samples at 37C for 10 minutes.TransformationTransformation1. Transform 1 l of each BP reaction into 50 l of One Shot OmniMAX 2 T1 Phage-Resistant Cells (Catalog no. C8540-03). Incubate on ice for 30 minutes. Heat-shock cells by incubating at 42C for 30 seconds. Add 250 l of S.O.C. Medium and incubate at 37C for 1 hour with shaking. Plate 20 l and 100 l of each transformation onto selective plates. Note: Any competent cells with a transformation efficiency of 1.0 10 8 transformants/g may be used. 2. Transform 1 l of pUC19 DNA (10 ng/ml) into 50 l of One Shot OmniMAX 2 T1 Phage-Resistant Cells as described above. Plate 20 l and 100 l on LB plates containing 100 g/ml kanamycin, or the appropriate selection marker for your donor vector.ExpectedExpected ResultsResults An efficient BP recombination reaction will produce 1500 colonies if the entire BP reaction is transformed and plated. LRLR ReactionReaction Transferring 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 room temperature and mix: Entry clone (50-150 ng) 1-7 l Destination vector (150 ng/l) 1 l TE buffer, pH 8.0 to 8 l 2. Thaw on ice the LR Clonase II enzyme mix for about 2 minutes. Vortex the LR Clonase II enzyme mix briefly twice (2 seconds each time). 3. To each sample (Step 1, above), add 2 l of LR Clonase II enzyme mix to the reaction and mix well by vortexing briefly twice. Microcentrifuge briefly. 4. Return LR Clonase II enzyme mix to -20C or -80C storage. 5. Incubate reactions at 25C for 1 hour. 6. Add 1 l of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly. Incubate samples at 37C for 10 minutes.TransformationTransformation Follow the protocol as indicated for the BP reaction, except use t