Opentrons Dual-Index Primer Plate Workflow v2

This protocol is designed to utilize the Opentrons robotic automation platform to generate dual-index primer - master mix plates for PCR reactions for NGS, particularly for DNA barcoding large specimen pools with Oxford Nanopore Technologies (ONT) MinION devices. It is designed for the indexing strategy where you start with a plate of 96 different forward or reverse primers, and then use a single forward/reverse index for each PRC plate you are running for the pool. I typically use eight plates (768 reactions) of specimens for a given Flongle run. So each plate of reactions has a single unique forward tag corresponding with that plate number and a standard plate of 96 reverse tags. The protocol is broken down into three primary areas. 1. OT-2 "Working" Primer Plates - 100uM->10uM These steps and automated protocol turns a single 100uM primer plate into four 10uM "working" primer plates. 2. OT-2 Dual-Index PCR Stock Plates This portion of the protocol will generate four "stock" dual-index primer / master mix plates. Each of these stock plates will generate 16 PCR ready plates for a single ONT single index (Ex - ONT01). This protocol will need to be repeated twice to create stock plates for each of the eight unique indexes for a run. 3. OT-2 Dual-Index PCR Ready Plates This protocol will create eight PCR ready plates from one PCR stock plate. Each cell has half-reactions (11.5uL; 12.5 total reaction volume with template) or quarter-reactions (5.7uL; 6.3uL total reaction volume). At the end of this step, you are left with dual indexed PCR plates that are ready for DNA template to be added and to be put in the thermocycler.