Cargo validation: barcode ligation

Protocol 5 of 7, and one of two ways to read every well back — do this one or Cargo validation: index PCR, not both. Comes after Cargo creation. Next is Library assembly in rounds.

Take the archived designs to clonal wells, mark each well so sequencing says which well it came from, and call a pass or a fail per well.

Have in hand

  • cargo archive plate one well a design, polyclonal, sealed and frozen
  • DMX barcode kit the lab's own barcoding plasmids, one group a picked plate

Leaves you with

  • clonal picked plate one well a picked colony, each well one clone
  • well calls a pass or a fail a well, and which design each well holds
0 of 6 steps done

Materials

NameSupplierCatalogueStoragePer runNoteSource
25 cm BioAssay plateCorning431111one spot a design50 µg/mL kanamycin; about 2,500 colonies a plate[1]
384-well culture plateBeckman Coulterc742901, one a 384 wells60 µL low-salt LB with 50 µg/mL kanamycin a well[1]
1536-well barcoding plateGreiner7822701, one a 4 picked plates[1]
DMX barcode kit-20 °C96 plasmids, 4 groups of 24the lab's own stock; its sequences are read from the copy you hold
Barcoding master mix-20 °C0.5 µL a wella well is 3.5 µL: 2 µL lysate, 0.5 µL barcodes, 0.5 µL water and 0.5 µL master mix[1]

Equipment: QPix XE Microbial Colony Picker, ECHO 525 acoustic liquid handler, Incubator at 37 °C, A sequencer, and a demultiplexer that can check an address

Plates

picked 1123456789101112131415161718192021222324ABCDEFGHIJKLMNOP
picked 1 384 wells · 3 kinds · Beckman Coulter #c74290 · one picked colony each in 60 µL low-salt LB with 50 µg/mL kanamycin · 288 of 384 wells picked, a quarter at a time
  • quarter 1
  • quarter 2
  • quarter 3
lysate 1123456789101112131415161718192021222324252627282930313233343536373839404142434445464748ABCDEFGHIJKLMNOPQRSTUVWXYZAAABACADAEAF
lysate 1 1536 wells · Greiner #782270 · 3.5 µL: 2 µL lysate, barcodes, water and mastermix · 4 picked plates to one

Array and pick

Expected result

  • One spot a design, at about 2,500 colonies a plate, which is the density picking wants.

Troubleshooting

A spot grows nothing
That design has no clone to read. Re-transform it from the archive before anyone re-synthesises it.

Notes

  • This run reads back every design, which a floor of zero does: 72 designs. The rest stay polyclonal and are never read one design at a time.
  • The archive is untouched: this reads a copy of it.

Expected result

  • 288 wells over 1 plate: 288 picked.
  • Every colony of one design sits on one plate.

Troubleshooting

A design in many fragments gives no clean colony
The chances above say which designs that is likeliest for. Pick more colonies of those designs before the plates are poured, not after.

Notes

  • 4 colonies a design is the only count measured: four gave a clean copy of 343 of 458 genes.[2]
  • 1 fragment: 40 designs, 100.0% of picks clean
  • 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.

Mark every well

Compress 1 picked plates into lysate 1 ECHO 525 acoustic liquid handler · 288 wells · picked 1 → lysate 1 · Invert the picked plates for 30 minutes first, so the cells gather at the meniscus[1]
FromToµL
picked 1 A1lysate 1 A12
picked 1 A3lysate 1 A22
picked 1 A5lysate 1 A32
picked 1 A7lysate 1 A42
picked 1 A9lysate 1 A52
picked 1 A11lysate 1 A62
picked 1 A13lysate 1 A72
picked 1 A15lysate 1 A82
picked 1 A17lysate 1 A92
picked 1 A19lysate 1 A102
picked 1 A21lysate 1 A112
picked 1 A23lysate 1 A122
picked 1 C1lysate 1 A132
picked 1 C3lysate 1 A142
picked 1 C5lysate 1 A152
picked 1 C7lysate 1 A162
picked 1 C9lysate 1 A172
picked 1 C11lysate 1 A182
picked 1 C13lysate 1 A192
picked 1 C15lysate 1 A202
picked 1 C17lysate 1 A212
picked 1 C19lysate 1 A222
picked 1 C21lysate 1 A232
picked 1 C23lysate 1 A242
picked 1 E1lysate 1 A252
picked 1 E3lysate 1 A262
picked 1 E5lysate 1 A272
picked 1 E7lysate 1 A282
picked 1 E9lysate 1 A292
picked 1 E11lysate 1 A302
picked 1 E13lysate 1 A312
picked 1 E15lysate 1 A322
picked 1 E17lysate 1 A332
picked 1 E19lysate 1 A342
picked 1 E21lysate 1 A352
picked 1 E23lysate 1 A362
picked 1 G1lysate 1 A372
picked 1 G3lysate 1 A382
picked 1 G5lysate 1 A392
picked 1 G7lysate 1 A402
picked 1 G9lysate 1 A412
picked 1 G11lysate 1 A422
picked 1 G13lysate 1 A432
picked 1 G15lysate 1 A442
picked 1 G17lysate 1 A452
picked 1 G19lysate 1 A462
picked 1 G21lysate 1 A472
picked 1 G23lysate 1 A482
picked 1 I1lysate 1 B12
picked 1 I3lysate 1 B22
picked 1 I5lysate 1 B32
picked 1 I7lysate 1 B42
picked 1 I9lysate 1 B52
picked 1 I11lysate 1 B62
picked 1 I13lysate 1 B72
picked 1 I15lysate 1 B82
picked 1 I17lysate 1 B92
picked 1 I19lysate 1 B102
picked 1 I21lysate 1 B112
picked 1 I23lysate 1 B122
picked 1 K1lysate 1 B132
picked 1 K3lysate 1 B142
picked 1 K5lysate 1 B152
picked 1 K7lysate 1 B162
picked 1 K9lysate 1 B172
picked 1 K11lysate 1 B182
picked 1 K13lysate 1 B192
picked 1 K15lysate 1 B202
picked 1 K17lysate 1 B212
picked 1 K19lysate 1 B222
picked 1 K21lysate 1 B232
picked 1 K23lysate 1 B242
picked 1 M1lysate 1 B252
picked 1 M3lysate 1 B262
picked 1 M5lysate 1 B272
picked 1 M7lysate 1 B282
picked 1 M9lysate 1 B292
picked 1 M11lysate 1 B302
picked 1 M13lysate 1 B312
picked 1 M15lysate 1 B322
picked 1 M17lysate 1 B332
picked 1 M19lysate 1 B342
picked 1 M21lysate 1 B352
picked 1 M23lysate 1 B362
picked 1 O1lysate 1 B372
picked 1 O3lysate 1 B382
picked 1 O5lysate 1 B392
picked 1 O7lysate 1 B402
picked 1 O9lysate 1 B412
picked 1 O11lysate 1 B422
picked 1 O13lysate 1 B432
picked 1 O15lysate 1 B442
picked 1 O17lysate 1 B452
picked 1 O19lysate 1 B462
picked 1 O21lysate 1 B472
picked 1 O23lysate 1 B482
picked 1 A2lysate 1 C12
picked 1 A4lysate 1 C22
picked 1 A6lysate 1 C32
picked 1 A8lysate 1 C42
picked 1 A10lysate 1 C52
picked 1 A12lysate 1 C62
picked 1 A14lysate 1 C72
picked 1 A16lysate 1 C82
picked 1 A18lysate 1 C92
picked 1 A20lysate 1 C102
picked 1 A22lysate 1 C112
picked 1 A24lysate 1 C122
picked 1 C2lysate 1 C132
picked 1 C4lysate 1 C142
picked 1 C6lysate 1 C152
picked 1 C8lysate 1 C162
picked 1 C10lysate 1 C172
picked 1 C12lysate 1 C182
picked 1 C14lysate 1 C192
picked 1 C16lysate 1 C202
picked 1 C18lysate 1 C212
picked 1 C20lysate 1 C222
picked 1 C22lysate 1 C232
picked 1 C24lysate 1 C242
picked 1 E2lysate 1 C252
picked 1 E4lysate 1 C262
picked 1 E6lysate 1 C272
picked 1 E8lysate 1 C282
picked 1 E10lysate 1 C292
picked 1 E12lysate 1 C302
picked 1 E14lysate 1 C312
picked 1 E16lysate 1 C322
picked 1 E18lysate 1 C332
picked 1 E20lysate 1 C342
picked 1 E22lysate 1 C352
picked 1 E24lysate 1 C362
picked 1 G2lysate 1 C372
picked 1 G4lysate 1 C382
picked 1 G6lysate 1 C392
picked 1 G8lysate 1 C402
picked 1 G10lysate 1 C412
picked 1 G12lysate 1 C422
picked 1 G14lysate 1 C432
picked 1 G16lysate 1 C442
picked 1 G18lysate 1 C452
picked 1 G20lysate 1 C462
picked 1 G22lysate 1 C472
picked 1 G24lysate 1 C482
picked 1 I2lysate 1 D12
picked 1 I4lysate 1 D22
picked 1 I6lysate 1 D32
picked 1 I8lysate 1 D42
picked 1 I10lysate 1 D52
picked 1 I12lysate 1 D62
picked 1 I14lysate 1 D72
picked 1 I16lysate 1 D82
picked 1 I18lysate 1 D92
picked 1 I20lysate 1 D102
picked 1 I22lysate 1 D112
picked 1 I24lysate 1 D122
picked 1 K2lysate 1 D132
picked 1 K4lysate 1 D142
picked 1 K6lysate 1 D152
picked 1 K8lysate 1 D162
picked 1 K10lysate 1 D172
picked 1 K12lysate 1 D182
picked 1 K14lysate 1 D192
picked 1 K16lysate 1 D202
picked 1 K18lysate 1 D212
picked 1 K20lysate 1 D222
picked 1 K22lysate 1 D232
picked 1 K24lysate 1 D242
picked 1 M2lysate 1 D252
picked 1 M4lysate 1 D262
picked 1 M6lysate 1 D272
picked 1 M8lysate 1 D282
picked 1 M10lysate 1 D292
picked 1 M12lysate 1 D302
picked 1 M14lysate 1 D312
picked 1 M16lysate 1 D322
picked 1 M18lysate 1 D332
picked 1 M20lysate 1 D342
picked 1 M22lysate 1 D352
picked 1 M24lysate 1 D362
picked 1 O2lysate 1 D372
picked 1 O4lysate 1 D382
picked 1 O6lysate 1 D392
picked 1 O8lysate 1 D402
picked 1 O10lysate 1 D412
picked 1 O12lysate 1 D422
picked 1 O14lysate 1 D432
picked 1 O16lysate 1 D442
picked 1 O18lysate 1 D452
picked 1 O20lysate 1 D462
picked 1 O22lysate 1 D472
picked 1 O24lysate 1 D482
picked 1 B1lysate 1 E12
picked 1 B3lysate 1 E22
picked 1 B5lysate 1 E32
picked 1 B7lysate 1 E42
picked 1 B9lysate 1 E52
picked 1 B11lysate 1 E62
picked 1 B13lysate 1 E72
picked 1 B15lysate 1 E82
picked 1 B17lysate 1 E92
picked 1 B19lysate 1 E102
picked 1 B21lysate 1 E112
picked 1 B23lysate 1 E122
picked 1 D1lysate 1 E132
picked 1 D3lysate 1 E142
picked 1 D5lysate 1 E152
picked 1 D7lysate 1 E162
picked 1 D9lysate 1 E172
picked 1 D11lysate 1 E182
picked 1 D13lysate 1 E192
picked 1 D15lysate 1 E202
picked 1 D17lysate 1 E212
picked 1 D19lysate 1 E222
picked 1 D21lysate 1 E232
picked 1 D23lysate 1 E242
picked 1 F1lysate 1 E252
picked 1 F3lysate 1 E262
picked 1 F5lysate 1 E272
picked 1 F7lysate 1 E282
picked 1 F9lysate 1 E292
picked 1 F11lysate 1 E302
picked 1 F13lysate 1 E312
picked 1 F15lysate 1 E322
picked 1 F17lysate 1 E332
picked 1 F19lysate 1 E342
picked 1 F21lysate 1 E352
picked 1 F23lysate 1 E362
picked 1 H1lysate 1 E372
picked 1 H3lysate 1 E382
picked 1 H5lysate 1 E392
picked 1 H7lysate 1 E402
picked 1 H9lysate 1 E412
picked 1 H11lysate 1 E422
picked 1 H13lysate 1 E432
picked 1 H15lysate 1 E442
picked 1 H17lysate 1 E452
picked 1 H19lysate 1 E462
picked 1 H21lysate 1 E472
picked 1 H23lysate 1 E482
picked 1 J1lysate 1 F12
picked 1 J3lysate 1 F22
picked 1 J5lysate 1 F32
picked 1 J7lysate 1 F42
picked 1 J9lysate 1 F52
picked 1 J11lysate 1 F62
picked 1 J13lysate 1 F72
picked 1 J15lysate 1 F82
picked 1 J17lysate 1 F92
picked 1 J19lysate 1 F102
picked 1 J21lysate 1 F112
picked 1 J23lysate 1 F122
picked 1 L1lysate 1 F132
picked 1 L3lysate 1 F142
picked 1 L5lysate 1 F152
picked 1 L7lysate 1 F162
picked 1 L9lysate 1 F172
picked 1 L11lysate 1 F182
picked 1 L13lysate 1 F192
picked 1 L15lysate 1 F202
picked 1 L17lysate 1 F212
picked 1 L19lysate 1 F222
picked 1 L21lysate 1 F232
picked 1 L23lysate 1 F242
picked 1 N1lysate 1 F252
picked 1 N3lysate 1 F262
picked 1 N5lysate 1 F272
picked 1 N7lysate 1 F282
picked 1 N9lysate 1 F292
picked 1 N11lysate 1 F302
picked 1 N13lysate 1 F312
picked 1 N15lysate 1 F322
picked 1 N17lysate 1 F332
picked 1 N19lysate 1 F342
picked 1 N21lysate 1 F352
picked 1 N23lysate 1 F362
picked 1 P1lysate 1 F372
picked 1 P3lysate 1 F382
picked 1 P5lysate 1 F392
picked 1 P7lysate 1 F402
picked 1 P9lysate 1 F412
picked 1 P11lysate 1 F422
picked 1 P13lysate 1 F432
picked 1 P15lysate 1 F442
picked 1 P17lysate 1 F452
picked 1 P19lysate 1 F462
picked 1 P21lysate 1 F472
picked 1 P23lysate 1 F482

Expected result

  • 288 wells of lysate, 4 picked plates to one.

3003504004506xHisT7 promoterRBSlac operatorinternal stufferPmeIPmeIXbaI (310)XmaI (385)SmaI - SrfI (387)DMX-iGGA N281 .. 490 (210 bp)
The cassette DMX-iGGA N carries, drawn empty: a picked well holds one with its design in place of the stuffer. The kit chains its 4 barcodes on, AGGA through TTCC, reading on the strand the cargo reads on.[3]
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Expected result

  • One barcoded construct a well, carrying four barcodes chained head to tail.

Notes

  • The address is worked out from where the well is, not looked up: three groups name the well and the fourth goes across the whole plate from a reservoir.
  • The kit's sequences are not shipped. Read them from the copy you hold.

Expected result

  • Reads for 288 wells, every well told from the rest by the marks it carries.

Notes

  • A pool saturates one miniprep column, which is why Qian's clean-up runs 2 columns.
  • Each pair starts at a different barcode, so every amplicon still carries the design and all four barcodes. Qian runs the pairs separately and pools what they make.
  • This read pools 1 plate. One axis of the address is the plate, so the marks tell 24 plates apart on one flow cell.

Call the wells

Expected result

  • A well read more than 150 times is called.
  • A pass matches across the whole designed region: both entry overhangs, the fragment, the stuffer and the barcode.

Troubleshooting

A well's call is the right protein and the wrong barcode
It fails. A barcode that no longer names its member cannot be put right by the linkage read later.

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.

Sources

  1. Qian et al. 2026, Supplementary Information · Nat. Commun. 10.1038/s41467-026-76740-9 · held under reference_docs/ · 2026-10-06 https://doi.org/10.1038/s41467-026-76740-9 cited at Day 2 · Day 3 · Day 4.1
  2. 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 · docs/research/synthesis-and-assembly.md https://doi.org/10.1021/acssynbio.3c00694 cited at colonies screened per gene
  3. liulab-mbio, Figure sources for four designs · docs/research/figure-sources.md cited at section 3, DMX