Solid-phase cloning for high-throughput assembly of single and multiple DNA parts [Elektronisk resurs]

• 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. 

Ämnesord

Natural Sciences  (hsv)

Biological Sciences  (hsv)

Naturvetenskap  (hsv)

Biologiska vetenskaper  (hsv)

Biotechnology  (kau)

Bioteknologi  (kau)

Indexterm och SAB-rubrik

PCR Products

Restriction Enzymes

Magnetic Beads

In-Vitro

One-Pot

Protein

Polymerase

Expression

Construction

Proteomics