{ "title": "AP-1 cargo read-back", "key": "781eedf6c51221ca", "summary": "Take 32 archived designs to 128 clonal wells, mark each well so sequencing says which well it came from, and call a pass or a fail per well.", "overview": { "Designs": "32 of 72 on the sheet", "Route": "index PCR", "Wells": "128 over 1 picked plate", "Selection": "kanamycin" }, "highlights": [ "A design is read back as a name and a count of the pieces it was built from, never as bases, so any clonal stock this lab holds can be read back here.", "The bench is sized from the 32 designs read and not from the sheet, so a design the floor leaves out costs no well, no plate and no reagent." ], "background": [ { "title": "Which designs are read back", "body": [ "This run reads every design built from 2 fragments or more: 32 of 72. The rest stay polyclonal and are never read one design at a time.", "Every design is spotted from AP-1 cargo archive plate, which this run reads a copy of and never changes. A design whose spot grows nothing has no clone to read, and is re-transformed before anyone re-synthesises it." ] }, { "title": "How a well is told from the rest", "body": [ "The index PCR route marks each well: amplify each well with one barcoded primer pair. The other route marks the same wells a different way, and the picking, the pass rule and the reformat are the same either way.", "A well's marks are worked out from where the well is rather than looked up, so a demultiplexer can check an address instead of trusting a file. One axis of the address is the plate: this run pools 2 plates, and the marks tell 96 plates apart on one flow cell.", "A well is called above 20 reads. The other route's floor is not a stricter or a looser setting of the same scale, so neither number carries over to the other route." ] } ], "checks": [], "choice": "", "consumes": [ { "name": "AP-1 cargo archive plate", "what": "one clonal stock a design, 72 of them, sealed and frozen", "spec": [], "storage": "-80 °C glycerol stock", "rules": [] }, { "name": "Barcoded index primer plate", "what": "the lab's index primer plate IDX-01, prepared once and called for by a run", "spec": [ "1 µM each" ], "storage": "-20 °C", "rules": [] } ], "produces": [ { "name": "clonal picked plate", "what": "one well a picked colony, each well one clone", "spec": [], "storage": "", "rules": [] }, { "name": "well calls", "what": "a pass or a fail a well, and which design each well holds", "spec": [], "storage": "", "rules": [] } ], "materials": [ { "name": "25 cm BioAssay plate", "supplier": "Corning", "catalog": "431111", "storage": "", "amount": "one spot a design", "note": "kanamycin; about 2,500 colonies a plate", "contains": [], "rules": [], "cautions": [], "citation": { "source": "Qian SI", "locator": "Day 2" } }, { "name": "384-well culture plate", "supplier": "Beckman Coulter", "catalog": "c74290", "storage": "", "amount": "1, one a 384 wells", "note": "60 µL low-salt LB with kanamycin a well", "contains": [], "rules": [], "cautions": [], "citation": { "source": "Qian SI", "locator": "Day 3" } }, { "name": "96-well half-skirted PCR plate", "supplier": "USA Scientific", "catalog": "1402-9700", "storage": "", "amount": "2, one a quarter of a picked plate", "note": "", "contains": [], "rules": [], "cautions": [], "citation": { "source": "LevSeq", "locator": "step 2" } }, { "name": "Barcoded index primer plate", "supplier": "", "catalog": "", "storage": "-20 °C", "amount": "2 µL a well at 1 µM each, 96 forward and 96 reverse", "note": "prepared once as lab stock, by its own protocol; a run calls for it. The SI's plate preparation ends at 0.1 µM, where its own protocol and the article both stamp 1 µM, which is what this takes", "contains": [], "rules": [], "cautions": [], "citation": { "source": "LevSeq", "locator": "step 3" } }, { "name": "ThermoPol Reaction Buffer", "supplier": "NEB", "catalog": "B9004", "storage": "-20 °C", "amount": "1 µL a reaction, 10X", "note": "", "contains": [], "rules": [], "cautions": [], "citation": { "source": "LevSeq", "locator": "step 1" } }, { "name": "dNTP mix", "supplier": "NEB", "catalog": "N0447", "storage": "-20 °C", "amount": "0.2 µL a reaction, 10 mM each", "note": "", "contains": [], "rules": [], "cautions": [], "citation": { "source": "LevSeq", "locator": "step 1" } }, { "name": "Taq DNA Polymerase", "supplier": "NEB", "catalog": "M0267", "storage": "-20 °C", "amount": "0.05 µL a reaction", "note": "5 U/µL, so one reaction takes 0.25 units", "contains": [], "rules": [], "cautions": [ "Keep the polymerase on ice." ], "citation": { "source": "PS-M0267", "locator": "Concentration" } }, { "name": "DMSO, molecular biology grade", "supplier": "MP Biomedicals", "catalog": "194819", "storage": "", "amount": "0.4 µL a reaction, 4% (v/v) in the well", "note": "", "contains": [], "rules": [], "cautions": [], "citation": { "source": "LevSeq", "locator": "step 1" } } ], "oligos": [], "order_sheet": "", "files": [], "equipment": [ "QPix XE Microbial Colony Picker", "multichannel pipette on 96-well spacing", "Thermocycler taking a 96-well plate", "Incubator at 37 °C", "A sequencer, and a demultiplexer that can check an address" ], "vessels": [], "plates": [ { "name": "picked 1", "wells": 384, "catalog": "Beckman Coulter #c74290", "holds": "one picked colony each in 60 µL low-salt LB with kanamycin", "seating": {}, "labels": { "A1": "quarter 1", "A3": "quarter 1", "A5": "quarter 1", "A7": "quarter 1", "A9": "quarter 1", "A11": "quarter 1", "A13": "quarter 1", "A15": "quarter 1", "A17": "quarter 1", "A19": "quarter 1", "A21": "quarter 1", "A23": "quarter 1", "C1": "quarter 1", "C3": "quarter 1", "C5": "quarter 1", "C7": "quarter 1", "C9": "quarter 1", "C11": "quarter 1", "C13": "quarter 1", "C15": "quarter 1", "C17": "quarter 1", "C19": "quarter 1", "C21": "quarter 1", "C23": "quarter 1", "E1": "quarter 1", "E3": "quarter 1", "E5": "quarter 1", "E7": "quarter 1", "E9": "quarter 1", "E11": "quarter 1", "E13": "quarter 1", "E15": "quarter 1", "E17": "quarter 1", "E19": "quarter 1", "E21": "quarter 1", "E23": "quarter 1", "G1": "quarter 1", "G3": "quarter 1", "G5": "quarter 1", "G7": "quarter 1", "G9": "quarter 1", "G11": "quarter 1", "G13": "quarter 1", "G15": "quarter 1", "G17": "quarter 1", "G19": "quarter 1", "G21": "quarter 1", "G23": "quarter 1", "I1": "quarter 1", "I3": "quarter 1", "I5": "quarter 1", "I7": "quarter 1", "I9": "quarter 1", "I11": "quarter 1", "I13": "quarter 1", "I15": "quarter 1", "I17": "quarter 1", "I19": "quarter 1", "I21": "quarter 1", "I23": "quarter 1", "K1": "quarter 1", "K3": "quarter 1", "K5": "quarter 1", "K7": "quarter 1", "K9": "quarter 1", "K11": "quarter 1", "K13": "quarter 1", "K15": "quarter 1", "K17": "quarter 1", "K19": "quarter 1", "K21": "quarter 1", "K23": "quarter 1", "M1": "quarter 1", "M3": "quarter 1", "M5": "quarter 1", "M7": "quarter 1", "M9": "quarter 1", "M11": "quarter 1", "M13": "quarter 1", "M15": "quarter 1", "M17": "quarter 1", "M19": "quarter 1", "M21": "quarter 1", "M23": "quarter 1", "O1": "quarter 1", "O3": "quarter 1", "O5": "quarter 1", "O7": "quarter 1", "O9": "quarter 1", "O11": "quarter 1", "O13": "quarter 1", "O15": "quarter 1", "O17": "quarter 1", "O19": "quarter 1", "O21": "quarter 1", "O23": "quarter 1", "A2": "quarter 2", "A4": "quarter 2", "A6": "quarter 2", "A8": "quarter 2", "A10": "quarter 2", "A12": "quarter 2", "A14": "quarter 2", "A16": "quarter 2", "A18": "quarter 2", "A20": "quarter 2", "A22": "quarter 2", "A24": "quarter 2", "C2": "quarter 2", "C4": "quarter 2", "C6": "quarter 2", "C8": "quarter 2", "C10": "quarter 2", "C12": "quarter 2", "C14": "quarter 2", "C16": "quarter 2", "C18": "quarter 2", "C20": "quarter 2", "C22": "quarter 2", "C24": "quarter 2", "E2": "quarter 2", "E4": "quarter 2", "E6": "quarter 2", "E8": "quarter 2", "E10": "quarter 2", "E12": "quarter 2", "E14": "quarter 2", "E16": "quarter 2" }, "rules": [], "note": "128 of 384 wells picked, a quarter at a time" }, { "name": "index 1", "wells": 96, "catalog": "USA Scientific #1402-9700", "holds": "one barcoded PCR each, the pair its own address names", "seating": {}, "labels": { "A1": "forward 1, reverse 1", "A2": "forward 2, reverse 1", "A3": "forward 3, reverse 1", "A4": "forward 4, reverse 1", "A5": "forward 5, reverse 1", "A6": "forward 6, reverse 1", "A7": "forward 7, reverse 1", "A8": "forward 8, reverse 1", "A9": "forward 9, reverse 1", "A10": "forward 10, reverse 1", "A11": "forward 11, reverse 1", "A12": "forward 12, reverse 1", "B1": "forward 13, reverse 1", "B2": "forward 14, reverse 1", "B3": "forward 15, reverse 1", "B4": "forward 16, reverse 1", "B5": "forward 17, reverse 1", "B6": "forward 18, reverse 1", "B7": "forward 19, reverse 1", "B8": "forward 20, reverse 1", "B9": "forward 21, reverse 1", "B10": "forward 22, reverse 1", "B11": "forward 23, reverse 1", "B12": "forward 24, reverse 1", "C1": "forward 25, reverse 1", "C2": "forward 26, reverse 1", "C3": "forward 27, reverse 1", "C4": "forward 28, reverse 1", "C5": "forward 29, reverse 1", "C6": "forward 30, reverse 1", "C7": "forward 31, reverse 1", "C8": "forward 32, reverse 1", "C9": "forward 33, reverse 1", "C10": "forward 34, reverse 1", "C11": "forward 35, reverse 1", "C12": "forward 36, reverse 1", "D1": "forward 37, reverse 1", "D2": "forward 38, reverse 1", "D3": "forward 39, reverse 1", "D4": "forward 40, reverse 1", "D5": "forward 41, reverse 1", "D6": "forward 42, reverse 1", "D7": "forward 43, reverse 1", "D8": "forward 44, reverse 1", "D9": "forward 45, reverse 1", "D10": "forward 46, reverse 1", "D11": "forward 47, reverse 1", "D12": "forward 48, reverse 1", "E1": "forward 49, reverse 1", "E2": "forward 50, reverse 1", "E3": "forward 51, reverse 1", "E4": "forward 52, reverse 1", "E5": "forward 53, reverse 1", "E6": "forward 54, reverse 1", "E7": "forward 55, reverse 1", "E8": "forward 56, reverse 1", "E9": "forward 57, reverse 1", "E10": "forward 58, reverse 1", "E11": "forward 59, reverse 1", "E12": "forward 60, reverse 1", "F1": "forward 61, reverse 1", "F2": "forward 62, reverse 1", "F3": "forward 63, reverse 1", "F4": "forward 64, reverse 1", "F5": "forward 65, reverse 1", "F6": "forward 66, reverse 1", "F7": "forward 67, reverse 1", "F8": "forward 68, reverse 1", "F9": "forward 69, reverse 1", "F10": "forward 70, reverse 1", "F11": "forward 71, reverse 1", "F12": "forward 72, reverse 1", "G1": "forward 73, reverse 1", "G2": "forward 74, reverse 1", "G3": "forward 75, reverse 1", "G4": "forward 76, reverse 1", "G5": "forward 77, reverse 1", "G6": "forward 78, reverse 1", "G7": "forward 79, reverse 1", "G8": "forward 80, reverse 1", "G9": "forward 81, reverse 1", "G10": "forward 82, reverse 1", "G11": "forward 83, reverse 1", "G12": "forward 84, reverse 1", "H1": "forward 85, reverse 1", "H2": "forward 86, reverse 1", "H3": "forward 87, reverse 1", "H4": "forward 88, reverse 1", "H5": "forward 89, reverse 1", "H6": "forward 90, reverse 1", "H7": "forward 91, reverse 1", "H8": "forward 92, reverse 1", "H9": "forward 93, reverse 1", "H10": "forward 94, reverse 1", "H11": "forward 95, reverse 1", "H12": "forward 96, reverse 1" }, "rules": [], "note": "plate mark 1; 96 of 96 wells used" }, { "name": "index 2", "wells": 96, "catalog": "USA Scientific #1402-9700", "holds": "one barcoded PCR each, the pair its own address names", "seating": {}, "labels": { "A1": "forward 1, reverse 2", "A2": "forward 2, reverse 2", "A3": "forward 3, reverse 2", "A4": "forward 4, reverse 2", "A5": "forward 5, reverse 2", "A6": "forward 6, reverse 2", "A7": "forward 7, reverse 2", "A8": "forward 8, reverse 2", "A9": "forward 9, reverse 2", "A10": "forward 10, reverse 2", "A11": "forward 11, reverse 2", "A12": "forward 12, reverse 2", "B1": "forward 13, reverse 2", "B2": "forward 14, reverse 2", "B3": "forward 15, reverse 2", "B4": "forward 16, reverse 2", "B5": "forward 17, reverse 2", "B6": "forward 18, reverse 2", "B7": "forward 19, reverse 2", "B8": "forward 20, reverse 2", "B9": "forward 21, reverse 2", "B10": "forward 22, reverse 2", "B11": "forward 23, reverse 2", "B12": "forward 24, reverse 2", "C1": "forward 25, reverse 2", "C2": "forward 26, reverse 2", "C3": "forward 27, reverse 2", "C4": "forward 28, reverse 2", "C5": "forward 29, reverse 2", "C6": "forward 30, reverse 2", "C7": "forward 31, reverse 2", "C8": "forward 32, reverse 2" }, "rules": [], "note": "plate mark 2; 32 of 96 wells used" } ], "steps": [ { "title": "Array 32 designs and grow", "key": "array-designs", "section": "Array and pick", "instructions": [ "Spot each design from its archive plate as its own spot on a 25 cm BioAssay plate.", "Grow overnight at 37 °C on kanamycin." ], "cautions": [], "notes": [ "This run reads back every design in 2 fragments or more: 32 designs. The rest stay polyclonal and are never read one design at a time.", "The archive is untouched: this reads a copy of it." ], "tables": [], "programs": [], "timers": [], "waits": [], "hands_on_seconds": null, "transfers": [], "figures": [], "gels": [], "expected": [ "One spot a design, at about 2,500 colonies a plate, which is the density picking wants." ], "calculator": null, "troubleshooting": [ { "problem": "A spot grows nothing", "solution": "That design has no clone to read. Re-transform it from the archive before anyone re-synthesises it.", "citation": null } ], "holes": [] }, { "title": "Pick 4 colonies of each design", "key": "pick-colonies", "section": "Array and pick", "instructions": [ "Pick 4 colonies a design into 60 µL low-salt LB with kanamycin, with the QPix XE Microbial Colony Picker.", "Fill one quarter of each 384-well plate before starting the next.", "Grow overnight at 37 °C." ], "cautions": [], "notes": [ { "text": "4 colonies a design is the only count measured: four gave a clean copy of 343 of 458 genes.", "citation": { "source": "lund-2024", "locator": "colonies screened per gene" } }, "2 fragments: 16 designs, 100.0% of picks clean", "3 fragments: 6 designs, 93.8% of picks clean", "4 fragments: 9 designs, 89.2% of picks clean", "5 fragments: 1 design, 84.6% of picks clean", "A quarter at a time is what makes a part-filled plate give full plates downstream, and what keeps a mark off a plate that is mostly empty." ], "tables": [], "programs": [], "timers": [], "waits": [], "hands_on_seconds": null, "transfers": [], "figures": [], "gels": [], "expected": [ "128 wells over 1 plate: 128 picked.", "Every colony of one design sits on one plate." ], "calculator": null, "troubleshooting": [ { "problem": "A design in many fragments gives no clean colony", "solution": "The chances above say which designs that is likeliest for. Pick more colonies of those designs before the plates are poured, not after.", "citation": null } ], "holes": [] }, { "title": "Sample the picked plates into index plates", "key": "sample-into-index-plates", "section": "Mark every well", "instructions": [ "Move 1 µL of each well into its 96-well plate, one quarter of the picked plate a pass." ], "cautions": [], "notes": [ "One well in four lines up under a head built for the smaller format, so a quarter moves in one pass." ], "tables": [], "programs": [], "timers": [], "waits": [], "hands_on_seconds": null, "transfers": [ { "title": "Sample a quarter of picked 1 into index 1", "moves": [ { "source": { "plate": "picked 1", "well": "A1" }, "destination": { "plate": "index 1", "well": "A1" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "A3" }, "destination": { "plate": "index 1", "well": "A2" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "A5" }, "destination": { "plate": "index 1", "well": "A3" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "A7" }, "destination": { "plate": "index 1", "well": "A4" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "A9" }, "destination": { "plate": "index 1", "well": "A5" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "A11" }, "destination": { "plate": "index 1", "well": "A6" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "A13" }, "destination": { "plate": "index 1", "well": "A7" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "A15" }, "destination": { "plate": "index 1", "well": "A8" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "A17" }, "destination": { "plate": "index 1", "well": "A9" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "A19" }, "destination": { "plate": "index 1", "well": "A10" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "A21" }, "destination": { "plate": "index 1", "well": "A11" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "A23" }, "destination": { "plate": "index 1", "well": "A12" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "C1" }, "destination": { "plate": "index 1", "well": "B1" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "C3" }, "destination": { "plate": "index 1", "well": "B2" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "C5" }, "destination": { "plate": "index 1", "well": "B3" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "C7" }, "destination": { "plate": "index 1", "well": "B4" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "C9" }, "destination": { "plate": "index 1", "well": "B5" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "C11" }, "destination": { "plate": "index 1", "well": "B6" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "C13" }, "destination": { "plate": "index 1", "well": "B7" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "C15" }, "destination": { "plate": "index 1", "well": "B8" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "C17" }, "destination": { "plate": "index 1", "well": "B9" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "C19" }, "destination": { "plate": "index 1", "well": "B10" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "C21" }, "destination": { "plate": "index 1", "well": "B11" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "C23" }, "destination": { "plate": "index 1", "well": "B12" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E1" }, "destination": { "plate": "index 1", "well": "C1" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E3" }, "destination": { "plate": "index 1", "well": "C2" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E5" }, "destination": { "plate": "index 1", "well": "C3" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E7" }, "destination": { "plate": "index 1", "well": "C4" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E9" }, "destination": { "plate": "index 1", "well": "C5" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E11" }, "destination": { "plate": "index 1", "well": "C6" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E13" }, "destination": { "plate": "index 1", "well": "C7" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E15" }, "destination": { "plate": "index 1", "well": "C8" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E17" }, "destination": { "plate": "index 1", "well": "C9" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E19" }, "destination": { "plate": "index 1", "well": "C10" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E21" }, "destination": { "plate": "index 1", "well": "C11" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E23" }, "destination": { "plate": "index 1", "well": "C12" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "G1" }, "destination": { "plate": "index 1", "well": "D1" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "G3" }, "destination": { "plate": "index 1", "well": "D2" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "G5" }, "destination": { "plate": "index 1", "well": "D3" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "G7" }, "destination": { "plate": "index 1", "well": "D4" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "G9" }, "destination": { "plate": "index 1", "well": "D5" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "G11" }, "destination": { "plate": "index 1", "well": "D6" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "G13" }, "destination": { "plate": "index 1", "well": "D7" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "G15" }, "destination": { "plate": "index 1", "well": "D8" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "G17" }, "destination": { "plate": "index 1", "well": "D9" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "G19" }, "destination": { "plate": "index 1", "well": "D10" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "G21" }, "destination": { "plate": "index 1", "well": "D11" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "G23" }, "destination": { "plate": "index 1", "well": "D12" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "I1" }, "destination": { "plate": "index 1", "well": "E1" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "I3" }, "destination": { "plate": "index 1", "well": "E2" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "I5" }, "destination": { "plate": "index 1", "well": "E3" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "I7" }, "destination": { "plate": "index 1", "well": "E4" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "I9" }, "destination": { "plate": "index 1", "well": "E5" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "I11" }, "destination": { "plate": "index 1", "well": "E6" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "I13" }, "destination": { "plate": "index 1", "well": "E7" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "I15" }, "destination": { "plate": "index 1", "well": "E8" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "I17" }, "destination": { "plate": "index 1", "well": "E9" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "I19" }, "destination": { "plate": "index 1", "well": "E10" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "I21" }, "destination": { "plate": "index 1", "well": "E11" }, 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"well": "C1" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E4" }, "destination": { "plate": "index 2", "well": "C2" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E6" }, "destination": { "plate": "index 2", "well": "C3" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E8" }, "destination": { "plate": "index 2", "well": "C4" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E10" }, "destination": { "plate": "index 2", "well": "C5" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E12" }, "destination": { "plate": "index 2", "well": "C6" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E14" }, "destination": { "plate": "index 2", "well": "C7" }, "volume_ul": 1.0 }, { "source": { "plate": "picked 1", "well": "E16" }, "destination": { "plate": "index 2", "well": "C8" }, "volume_ul": 1.0 } ], "instrument": "multichannel pipette", "note": "one well in four, which is the spacing a head built for the index format reads", "citation": { "source": "LevSeq", "locator": "step 4" } } ], "figures": [], "gels": [], "expected": [ "2 index plates, 128 reactions in all." ], "calculator": null, "troubleshooting": [], "holes": [] }, { "title": "Amplify each well with its own pair", "key": "index-pcr", "section": "Mark every well", "instructions": [ "Add 7 µL of the master mix below to each well, which already holds its 1 µL of culture.", "Add 2 µL of the pair its address names from the lab's index primer plate IDX-01, for 10 µL a well.", "Seal the plate, spin it down, and run the program below." ], "cautions": [], "notes": [ "96 forward marks and 96 reverse reach 9,216 wells, so the pairs already held cover far more than this run needs.", "The primer plate is built once as lab stock and a run calls for it; this protocol does not build one.", "The first 10 cycles touch down from 68 to 63.5 °C, 0.5 °C a cycle, and 25 more run at 68 °C.", "Extension is 2 minutes, what the authors ran for a gene below 1 kb. The published rule is at least one minute a kilobase, so a longer design wants it raised.", "The mix is made 1.5 times what the wells take, which is what LevSeq's own per-plate table spells out. Nothing says what the surplus is for." ], "tables": [ { "components": [ { "name": "ThermoPol Reaction Buffer", "volume_ul": 1.0, "stock": "10X", "final": "1X", "master_mix": true, "citation": { "source": "LevSeq", "locator": "step 1" }, "calculator": null }, { "name": "dNTP mix", "volume_ul": 0.2, "stock": "10 mM each", "final": "0.2 mM each", "master_mix": true, "citation": { "source": "LevSeq", "locator": "step 1" }, "calculator": null }, { "name": "Taq DNA Polymerase", "volume_ul": 0.05, "stock": "5 U/µL", "final": "0.25 units", "master_mix": true, "citation": { "source": "LevSeq", "locator": "step 1" }, "calculator": null }, { "name": "DMSO", "volume_ul": 0.4, "stock": "100%", "final": "4% (v/v)", "master_mix": true, "citation": { "source": "LevSeq", "locator": "step 1" }, "calculator": null }, { "name": "Nuclease-free water", "volume_ul": 5.35, "stock": "", "final": "", "master_mix": true, "citation": { "source": "LevSeq", "locator": "step 1" }, "calculator": null }, { "name": "Barcoded primer mix", "volume_ul": 2.0, "stock": "1 µM each", "final": "0.2 µM each", "master_mix": false, "citation": { "source": "LevSeq", "locator": "step 3" }, "calculator": null }, { "name": "Overnight culture", "volume_ul": 1.0, "stock": "", "final": "", "master_mix": false, "citation": { "source": "LevSeq", "locator": "step 4" }, "calculator": null } ], "title": "Index PCR, one well a sample", "reactions": 128, "overage": 0.5 } ], "programs": [ { "stages": [ { "incubations": [ { "label": "Initial denaturation", "temperature_c": 95.0, "seconds": 300, "delta_c": null, "citation": { "source": "LevSeq", "locator": "thermal cycler table" } } ], "cycles": 1, "citation": null }, { "incubations": [ { "label": "Denature", "temperature_c": 95.0, "seconds": 20, "delta_c": null, "citation": { "source": "LevSeq", "locator": "thermal cycler table" } }, { "label": "Anneal", "temperature_c": 68.0, "seconds": 20, "delta_c": -0.5, "citation": { "source": "LevSeq", "locator": "thermal cycler table" } }, { "label": "Extend", "temperature_c": 68.0, "seconds": 120, "delta_c": null, "citation": { "source": "LevSeq", "locator": "thermal cycler table" } } ], "cycles": 10, "citation": null }, { "incubations": [ { "label": "Denature", "temperature_c": 95.0, "seconds": 20, "delta_c": null, "citation": { "source": "LevSeq", "locator": "thermal cycler table" } }, { "label": "Anneal and extend", "temperature_c": 68.0, "seconds": 120, "delta_c": null, "citation": { "source": "LevSeq", "locator": "thermal cycler table" } } ], "cycles": 25, "citation": null }, { "incubations": [ { "label": "Final extension", "temperature_c": 68.0, "seconds": 300, "delta_c": null, "citation": { "source": "LevSeq", "locator": "thermal cycler table" } } ], "cycles": 1, "citation": null }, { "incubations": [ { "label": "Hold", "temperature_c": 4.0, "seconds": null, "delta_c": null, "citation": { "source": "LevSeq", "locator": "thermal cycler table" } } ], "cycles": 1, "citation": null } ], "title": "Index PCR", "lid_temperature_c": null } ], "timers": [], "waits": [], "hands_on_seconds": null, "transfers": [], "figures": [], "gels": [], "expected": [ "One barcoded amplicon a well. A well's own place in the plate names its forward mark — the first well the first mark — and every well of one plate takes that plate's own reverse mark.", "One band a well, the design plus about 100 bp: the two marks, and the stretch between the primer sites and the reading frame." ], "calculator": null, "troubleshooting": [ { "problem": "A plate reads back far fewer wells than the others", "solution": "LevSeq traces that to the amplification, not the sequencing: on a run of ten plates three returned under 60% of their variants. Check each pool on a gel before the library prep.", "citation": null } ], "holes": [] }, { "title": "Pool and sequence", "key": "pool-and-sequence", "section": "Mark every well", "instructions": [ "Pool each index plate on its own and clean the pool up.", "Sequence the pool." ], "cautions": [], "notes": [ "This read pools 2 plates. One axis of the address is the plate, so the marks tell 96 plates apart on one flow cell." ], "tables": [], "programs": [], "timers": [], "waits": [], "hands_on_seconds": null, "transfers": [], "figures": [], "gels": [], "expected": [ "Reads for 128 wells, every well told from the rest by the marks it carries." ], "calculator": null, "troubleshooting": [], "holes": [] }, { "title": "Call every well", "key": "call-wells", "section": "Call the wells", "instructions": [ "Demultiplex the reads by address, checking each address rather than trusting a file.", "Call a consensus a well, then compare it with that well's design base for base.", "Reformat, compacting out the wells that failed." ], "cautions": [], "notes": [ "A well read too thinly carries no verdict and is not a failure, so reformatting does not compact it out.", "More than one consensus is mixed, and mixed fails.", "A design with no passing well is picked again from the same archive spot before anyone re-synthesises it." ], "tables": [], "programs": [], "timers": [], "waits": [], "hands_on_seconds": null, "transfers": [], "figures": [], "gels": [], "expected": [ "A well read more than 20 times is called; the index PCR route tolerates 10 reads and warns between the two.", "A pass matches across the whole designed region: both entry overhangs, the fragment, the stuffer and the barcode." ], "calculator": null, "troubleshooting": [ { "problem": "A well's call is the right protein and the wrong barcode", "solution": "It fails. A barcode that no longer names its member cannot be put right by the linkage read later.", "citation": null } ], "holes": [] } ], "sources": { "Qian SI": { "document": "Qian et al. 2026, Supplementary Information", "edition": "Nat. Commun. 10.1038/s41467-026-76740-9", "url": "https://doi.org/10.1038/s41467-026-76740-9", "read_as": "held under reference_docs/", "date": "2026-10-06", "note": "" }, "LevSeq": { "document": "Long, Y. et al. 2025, LevSeq, Supporting Information", "edition": "", "url": "https://doi.org/10.1021/acssynbio.4c00625", "read_as": "held under reference_docs/", "date": "2026-10-06", "note": "" }, "lund-2024": { "document": "Lund, S. et al. (2024) Highly parallelized construction of DNA from low-cost oligonucleotide mixtures using Data-optimized Assembly Design and Golden Gate. ACS Synth. Biol. 13, 745-751", "edition": "", "url": "https://doi.org/10.1021/acssynbio.3c00694", "read_as": "", "date": "", "note": "docs/research/synthesis-and-assembly.md" }, "PS-M0267": { "document": "New England Biolabs, Product Specification: Taq DNA Polymerase with ThermoPol Buffer", "edition": "PS-M0267S/L/X/E v2.0, effective 12 Feb 2020", "url": "https://www.neb.com/en/-/media/catalog/specifications/m/0/m0267s_l_x_e_v2.pdf", "read_as": "plain curl", "date": "2026-10-08", "note": "docs/research/route-b-index-pcr.md" } }, "holes": [], "bill": null }