Journal: Nucleic Acids Research
Article Title: Solid-phase cloning for high-throughput assembly of single and multiple DNA parts
Figure Lengend Snippet: Results from head-to-tail SPC. ( A ) Comparison of compatibility of thermostable polymerases with the head-to-tail method. The polymerases with best proof-reading capabilities, Phusion and Deep Vent, all failed to generate transformants with the standard head-to-tail protocol. ( B ) Activity was retained for protocols using Phusion after supplementation of the washing buffer with SDS. ( C ) Colony screens of inserted region representing a selection of assemblies of various lengths and number of inserts assembled by head-to-tail SPC. Final construct sizes spanned 2.9 to 7.2 kbps.
Article Snippet: DNA parts were amplified with Phusion® Hot-Start Flex (2 U/μl, New England Biolabs, Ipswich, MA, USA) by the following PCR program: 98°C for 30 s, 30 cycles of 98°C 8 s, 25 s annealing with temperature depending on primer melting temperature and 72°C for 20 s/kb, before ending with 72°C for 7 min followed by 4°C hold.
Techniques: Activity Assay, Selection, Construct