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- W4205276196 abstract "GateWay recombination cloning is achieved by flanking your gene of interest with GateWay attachment sites. In our case attB1 and attB2. Those sites are added to the PCR product via primers with 5' extensions. Since those primes create 31 bp and 30 bp 5' primer extensions respectively, plus about 20 bp of actual binding primer sequence it becomes expensive fast if you need 2 x ~50 bp primers for every GOI. We therefore use a 2 step PCR process to attach GateWay attB1 and attB2 sites. We first run a gene specific PCR with primers carrying short 5' extesions, and then a second PCR utilizing universal GateWay primers which bind to the short extension of the first PCR product to create the full attB1 and attB2 sites. This protocol has been adapted from: 2-STEP GATEWAY PCR EXPERIMENTS" @default.
- W4205276196 created "2022-01-25" @default.
- W4205276196 creator A5088221195 @default.
- W4205276196 date "2022-01-10" @default.
- W4205276196 modified "2023-10-15" @default.
- W4205276196 title "Preparing Gene of interest for GateWay cloning (2 step PCR process) v2" @default.
- W4205276196 doi "https://doi.org/10.17504/protocols.io.b3muqk6w" @default.
- W4205276196 hasPublicationYear "2022" @default.
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