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Lundqvist M, Edfors F, Sivertsson Å, Hallström BM, Hudson EP, Tegel H, Holmberg A, Uhlén M, Rockberg J
Nucleic Acids Res. 43 (7) e49 [2015-04-20; online 2015-01-23]
We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.
PubMed 25618848
DOI 10.1093/nar/gkv036
Crossref 10.1093/nar/gkv036
pii: gkv036
pmc: PMC4402512