Part carrier

Protocol 1 of 7. The run starts here. Next is Primer plates.

Put each of 72 parts in its own pCR-Blunt II-TOPO, one part a well, so the collection keeps and BsmBI can release any of them again.

parts to seat one part a tube, blunt and flanked so BsmBI releases it from its carrier

part carrier plate one pCR-Blunt II-TOPO a part, one part a well, selected on KanR

Materials

Name	Storage	Per run	Note	Source
pCR-Blunt II-TOPO	-20 °C		the part carrier; its backbone carries no BsmBI site, which is what lets BsmBI release the part again later and leave the backbone whole
Zero Blunt TOPO PCR Cloning Kit	-20 °C	one reaction per well	supplies the carrier already linearised with topoisomerase I bound to each 3' end, so the reaction adds no ligase and no other enzyme	^[1]

Part carrier plate 96 wells · 72 kinds · one seated part each, selected on KanR · 72 of 96 wells used

Set up one Zero Blunt TOPO PCR Cloning Kit reaction per well of Part carrier plate, each holding one blunt part, the linearised carrier and the kit's salt solution.
Leave 5 min at room temperature. Topoisomerase I comes bound to the carrier, so nothing is added to join the two.
Transform each well on its own and select on KanR.

TOPO reaction

72 carrier plasmids, one a part, each still named by the well it sits in.
No pool and no library: nothing here is mixed, so nothing has to be told apart afterwards.

Two parts end up in one well: The wells were pooled. One part a well is on purpose: a part that has met another cannot be re-cut back out on its own.

BsmBI releases a part from its carrier afterwards. The backbone carries no site of it, so a part comes back out on its own and the plasmid stays whole.

Thermo Fisher Scientific, Zero Blunt TOPO PCR Cloning Kit user guide · docs/research/synthesis-and-assembly.md cited at TOPO cloning reaction