Preparing Gene of interest for GateWay cloning (2 step PCR process) v2

• DOI: 10.17504/protocols.io.b3muqk6w
• Publication Date: 2022-01-10T04:09:12Z
• Authors: Johannes Wolfram ...
• Keywords: Primer (cosmetics); Gateway (web page); In silico PCR; Cloning (programming)

GateWay recombination cloning is achieved by flanking your gene of interest with attachment sites. In our case attB1 and attB2. Those sites are added to the PCR product via primers 5' extensions. Since those primes create 31 bp 30 primer extensions respectively, plus about 20 actual binding sequence it becomes expensive fast if you need 2 x ~50 for every GOI. We therefore use a step process attach attB2 first run specific carrying short extesions, then second utilizing universal which bind extension full This protocol has been adapted from: 2-STEP GATEWAY EXPERIMENTS