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Protocol ?for ? Single ?step ?BP/LR ?combined ?Gateway? ?reactions ? Xiquan ?Liang, ?Lansha ?Peng, ?Chang-?\Ho ?Baek, ?and ?Federico ?Katzen ? Life ?Technologies, ?Carlsbad, ?CA ?92008 ? Reagents ? 1.

Gateway? ?LR ?Clonase? ?II ?Enzyme ?mix ?(Life ?Technologies, ?Carlsbad, ?CA, ?US) ? 2. Gateway? ?BP ?Clonase? ?II ?Enzyme ?mix ?(Life ?Technologies, ?Carlsbad, ?CA, ?US) ? 3. pDONR?221 ?(Life ?Technologies, ?Carlsbad, ?CA, ?US) ?or ?any ?other ?Gateway donor ?vector ? 4. Gateway destination ?vector ? 5. A ?linear ?DNA ?fragment ?flanked ?by ?attB1 ?and ?attB2 ?recombination ?sequences ?or ?a ?Gateway expression ?vector. ? 6. TE ?buffer, ?pH ?8.0 ? 7. Any ?competent ?cells ?with ?a ?transformation ?efficiency ?of ?>1.0

108 ?transformants/?g ?and ?sensitive ?to ? the ?effects ?of ?the ?ccdB ?gene ?may ?be ?used ?(e.g. ?DH5α?, ?TOP10, ?DH10B?, ?OmniMAX?) ? 8. LB ?agar ?plates ?supplemented ?with ?the ?corresponding ?antibiotics. ? Procedures ? Protocol ?1 ?(to ?obtain ?numerous ?colonies ?bearing ?expression ?clones ?and ?limited ?number ?of ?colonies ? containing ?entry ?clones): ? 1. Add ?the ?following ?components ?to ?a ?1.5 ?ml ?microcentrifuge ?tube ?at ?room ?temperature ?and ?mix: ? attB-?\PCR ?fragment ?or ?expression ?clone ?(100 ?ng) ?1-?\6 ??l ? ? ? Donor ?vector ?(150 ?ng/?l) ?1-?\6 ??l ? ? Destination ?vector ?(150 ?ng/?l) ?1-?\6 ??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 ?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. Incubate ?reactions ?at ?25°C ?or ?at ?room ?temperature ?for ?1 ?to ?3 ?hours. 5. Add ?1 ??l ?of ?the ?Proteinase ?K ?solution ?to ?each ?sample ?to ?terminate ?the ?reaction. ?Vortex ?briefly. ? Incubate ?samples ?at ?37°C ?for ?10 ?minutes. ? 6. Transform ?chemically ?or ?electrocompetent ?cells ?following ?the ?corresponding ?guidelines ? 7. Plate ?on ?LB ?agar ?plates ?supplemented ?with ?the ?corresponding ?antibiotics ?(usually ?ampicillin). ? ? Protocol ?2 ?(to ?obtain ?numerous ?colonies ?containing ?entry ?and ?expression ?clones): ? 1. Add ?the ?following ?components ?to ?a ?1.5 ?ml ?microcentrifuge ?tube ?at ?room ?temperature ?and ?mix: ? attB-?\PCR ?fragment ?or ?expression ?clone ?(100 ?ng) ?1-?\4 ??l ? ? ? Donor ?vector ?(150 ?ng) ?1-?\4 ??l ? ? Destination ?vector ?(150 ?ng) ?1-?\4 ??l ? ? TE ?buffer, ?pH ?8.0 ?to ?6 ??l ? ? 2. Thaw ?on ?ice ?the ?LR ?Clonase? ?II ?and ?BP ?Clonase? ?II ?enzyme ?mixes ?for ?about ?2 ?minutes. ?Vortex ?the ? mixes ?briefly ?twice ?(2 ?seconds ?each ?time) 3. To ?each ?sample ?(Step ?1, ?above), ?add ?3 ??l ?of ?LR ?Clonase? ?II ?enzyme ?mix ?and ?1 ??l ?of ?BP ?Clonase? ?II ? enzyme ?mix ?to ?the ?reaction ?and ?mix ?well ?by ?vortexing ?briefly ?twice. ?Microcentrifuge ?briefly. 4. Incubate ?reactions ?at ?25°C ?or ?at ?room ?temperature ?for ?1 ?to ?3 ?hours. 5. Add ?1 ??l ?of ?the ?Proteinase ?K ?solution ?to ?each ?sample ?to ?terminate ?the ?reaction. ?Vortex ?briefly. ? Incubate ?samples ?at ?37°C ?for ?10 ?minutes. ? 6. Transform ?chemically ?or ?electrocompetent ?cells ?following ?the ?corresponding ?guidelines ? 7. Plate ?on ?two ?types ?of ?LB ?agar ?plates ?supplemented ?with ?the ?corresponding ?antibiotics ?(usually ? ampicillin ?to ?select ?for ?the ?expression ?clone, ?and ?kanamycin ?to ?select ?for ?the ?entry ?clone). ?