Cargo creation

Protocol 4 of 7. Comes after Cargo ordering and pool preparation. Next is one of the two ways to read every well back: Cargo validation: barcode ligation or Cargo validation: index PCR.

Pull every block out of the ordered pool and close each one's cargo into the vector for the position it fills, one archived well a design.

Have in hand

oligo pool the pool resuspended
primer working plate 1 a copy of primer stock plate, used for one run · 10 µM, 100 µL per well
block vector 1 DMX-iGGA N, ready to open
block vector 2 DMX-iGGA DBD, ready to open
block vector 3 DMX-iGGA C, ready to open

Leaves you with

cargo archive plate one well a design, polyclonal, sealed and frozen

primer working plate 1: never return to the freezer a thawed working plate is used once and discarded

2 numbers in this protocol have no source.

Materials

Name	Supplier	Catalogue	Storage	Per run	Note	Source
Q5 DNA Polymerase	NEB	M0491	-20 °C	one reaction per batch, then one per block	the buffer the annealing temperatures below were computed in
Pool amplification primers			-20 °C	74 primers: 1 P1 batch forward, 72 P2 gene reverse, 1 P3 batch outer	ordered from pool-primers.tsv; a pair a batch, an inner primer a block
NEBridge Golden Gate Assembly Kit, BsmBI-v2	NEB	E1602	-20 °C	one assembly per block	its mix carries the BsmBI that cuts every oligo back to its own fragment, which this method reserves, and the T4 DNA Ligase that joins them
dNTP mix	NEB	N0447	-20 °C	0.2 µL a reaction, 10 mM each		^[1]
DMX-iGGA vector			-20 °C	3, one a position	opened by the internal digest in round 1, and to take each block's cargo. A part enters on its own position's entry overhang, so there is one a position: DMX-iGGA N (block-vector-1.dna), DMX-iGGA DBD (block-vector-2.dna) and DMX-iGGA C (block-vector-3.dna)
BbsI-HF	New England Biolabs	R3539	-20 °C	2.5 µL per digest	opens the library, excising its stuffer
SrfI	New England Biolabs	R0629	-20 °C	2.5 µL per digest	cuts the excised stuffer, so it cannot ligate back
CutSmart Buffer			-20 °C		both digests run in it
SPRI paramagnetic beads				2 volumes after a digest, 1 after a ligation	the two ratios differ; both elute in water
Endura ElectroCompetent Cells	Lucigen	60242-2	-80 °C	4 aliquots, one a pulse, 25 µL each	pulsed at 1800 V, 600 Ω and 10 µF in a 1 mm cuvette	^[2]
Recovery medium				one outgrowth per round
Selective broth and plates					LB with 50 µg/mL kanamycin, the destination vector's own marker (KanR)

Equipment: Thermocycler taking a 96-well plate, Gel tank and a transilluminator

Caution: Keep the polymerase on ice.

Caution: Keep the cells and the cuvette on ice; a warm cuvette arcs.

Plates

PCR2 plate 96 wells · 72 kinds · one block a well, in the order its batch allotted the inner primers · 1 plate, one a batch; batch 1 holds 72

Amplify the pool

1PCR1: pull 1 batch out of the pool

Caution: Keep the polymerase on ice.

Set up 1 reaction, one a batch, with the pool as template.
Give each its own pair: batch 1: OP107 with OP146.
Run the program below. On a real-time instrument, add an intercalating dye and stop before the curve plateaus; the printed count is what to run without one.

PCR1, one tube a batch

Reactions mix includes 10% extra

Component	Stock	Final	1 rxn (µL)	Mix for 1 (µL)
Q5 Reaction Buffer	5X	1X	10	11
dNTP mix	10 mM each	200 µM each	1	1.1
Forward primer	10 µM	500 nM	2.5	2.75
Reverse primer	10 µM	500 nM	2.5	2.75
Template DNA			1	each tube
Q5 DNA Polymerase	2 U/µL	1 unit	0.5	0.55
Nuclease-free water		to 50 µL	32.5	35.8
Total			50	53.9 mix only

Put 49 µL of mix in each tube, then add 1 µL Template DNA.

PCR1 · 12 min 30 s plus ramps

Step	Temperature	Time	Cycles
Initial denaturation	98 °C	30 s	1
Denature	98 °C	10 s	×12^[3]
Anneal	65.1 °C	20 s
Extend	72 °C	20 s
Final extension	72 °C	2 min	1
Hold	4 °C	∞	1

PCR1

H29 no sourced number — PCR1, the polymerase the cycle count is stated against: no cycle count published for Q5 on a Twist oligo pool. Waiting on a number nobody has published. Filled by a count Twist states against Q5, or a pilot on this pool.

PCR1 pairs OP107 with OP146 and pulls one batch out of the pool, in 1 reaction^[4]

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Expected result

One band at 350 bp in every batch, which is the whole oligo.
Batch sizes: 1 holds 72.

PCR1, any batch

L NEB 100 bp DNA Ladder (N3231)
1 PCR1 product: 350 bp

Troubleshooting

No band: Drop the annealing temperature by 3 °C and check the pool went in.
A hump after the peak on a Bioanalyzer or TapeStation trace: Heteroduplexes, which is what over-amplification leaves. Run it again with fewer cycles. What too many cycles cost is dropout, chimeras and polymerase error; pooling the blocks equimolar later restores the evenness but none of those.

Notes

Every pair anneals at 65.1 °C, so one block of tubes takes them all.
The band for a 350 nt pool is 12 to 14 cycles, and more cycles give worse uniformity, so 12 is what prints.^[5]
The outer primer is what makes a batch a batch: it is dropped at PCR2, so a block cannot be pulled out of a batch it does not sit in.

2PCR2: pull each of the 72 blocks out of its batch

Caution: Keep the polymerase on ice.

Set up one reaction a block, 72 in all, in PCR2 plate.
Give each its batch's PCR1 product as template, that batch's forward primer, and the block's own inner primer.
Run the program below. Nobody published a cycle count for this reaction, so run it on a real-time instrument with an intercalating dye and stop before the curve plateaus.

PCR2, one well a block

Reactions mix includes 10% extra

Component	Stock	Final	1 rxn (µL)	Mix for 72 (µL)
Q5 Reaction Buffer	5X	1X	10	792
dNTP mix	10 mM each	200 µM each	1	79.2
Forward primer	10 µM	500 nM	2.5	198
Reverse primer	10 µM	500 nM	2.5	198
Template DNA			1	each tube
Q5 DNA Polymerase	2 U/µL	1 unit	0.5	39.6
Nuclease-free water		to 50 µL	32.5	2,574
Total			50	3,881 mix only

Put 49 µL of mix in each tube, then add 1 µL Template DNA.

PCR2

Step	Temperature	Time	Cycles
Initial denaturation	98 °C	30 s	1
Denature	98 °C	10 s	no sourced number
Anneal	64.3 °C	20 s
Extend	72 °C	20 s
Final extension	72 °C	2 min	1
Hold	4 °C	∞	1

H30 no sourced number — PCR2, the cycle count: no cycle count for PCR2, which pulls one block out of an already-amplified batch. Waiting on a number nobody has published. Filled by a pilot titrated against the heteroduplex hump on capillary electrophoresis, or a real-time run stopped before the curve plateaus.

PCR2 pairs OP107 with OP1 and pulls one block out of its batch, in 72 reactions^[4]

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Expected result

One band at 330 bp in every well, the outer primer's 20 nt shorter than PCR1's.
72 wells filled across 1 plate.

PCR2, any well

L NEB 100 bp DNA Ladder (N3231)
1 PCR2 product: 330 bp

Troubleshooting

A band at PCR1's length: The outer primer region was not dropped. Check the inner primer went in, and that the template is PCR1's product and not the pool.
A hump after the peak on a Bioanalyzer or TapeStation trace: Heteroduplexes, which is what over-amplification leaves. Run it again with fewer cycles; the forward primer is the whole batch's, so over-cycling here also pulls a neighbour's product into the well.

Notes

The pairs anneal between 64.3 and 65.1 °C. The program runs at 64.3, the lowest of them, so one block of tubes takes them all.
A block is named by its well from here on, not by anything in the tube.

Close each cargo into its vector

3Assemble each cargo into its position's destination, from its 1 to 5 pieces

Open DMX-iGGA N (block-vector-1.dna), DMX-iGGA DBD (block-vector-2.dna) and DMX-iGGA C (block-vector-3.dna) with BbsI and SrfI, the digest a round opens the library with, and clean each up.
Set up one assembly a well, holding that block's own PCR2 pieces, the opened destination of the position that block fills, and nothing from another well.
Transform, plate on 50 µg/mL kanamycin, and pick one colony a block.

Golden Gate assembly, NEBridge kit E1602

Reactions mix includes 10% extra

Component	Stock	Final	1 rxn (µL)	Mix for 72 (µL)
DMX-iGGA, opened^[6]		0.05 pmol (165.17 ng)	1	each tube
PCR2 piece 1^[6]		0.05 pmol (10.16 ng)	1	each tube
PCR2 piece 2^[6]		0.05 pmol (10.16 ng)	1	each tube
PCR2 piece 3^[6]		0.05 pmol (10.16 ng)	1	each tube
PCR2 piece 4^[6]		0.05 pmol (10.16 ng)	1	each tube
PCR2 piece 5^[6]		0.05 pmol (10.16 ng)	1	each tube
T4 DNA Ligase Buffer^[7]	10X	1X	2	158
NEBridge Golden Gate Enzyme Mix^[7]			1	79.2
Nuclease-free water		to 20 µL	11	871
Total			20	1,109 mix only

Put 14 µL of mix in each tube, then add 1 µL DMX-iGGA, opened, 1 µL PCR2 piece 1, 1 µL PCR2 piece 2, 1 µL PCR2 piece 3, 1 µL PCR2 piece 4, 1 µL PCR2 piece 5.

Golden Gate assembly · 1 h 5 min plus ramps

Step	Temperature	Time	Cycles
Digest	42 °C	1 min	×30^[7]
Ligate	16 °C	1 min	×30^[7]
End soak	60 °C	5 min	1^[7]

Golden Gate assembly

The stuffer BbsI cuts out of DMX-iGGA N, which a cargo replaces. The cargo enters on the four bases its position spells here.^[4]

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Expected result

72 plasmids, one a block: its position's destination carrying that block's cargo, 83 to 1,099 bp of it.
Each cargo reads: the overhang its part enters on, its coding bases, the internal stuffer, its barcode, and the TTCC cloning scar. The external stuffers parts.tsv spells either side of it are the destination's own bases.

Troubleshooting

A block comes out short: A piece was missing from the well. Check that well's PCR2 lane before assembling it again; the pieces of one block are not interchangeable.
Colonies carrying the destination with no cargo: The destination was not opened to completion. Run a little of its digest on a gel before setting the plate up again.

Notes

BsmBI cuts each oligo back to its fragment, so the primer sites, the recognition sites and the padding all stay outside the cargo.
The table is sized at the most pieces any block takes, so one master mix covers the plate; a well with fewer pieces fills fewer of its DNA rows.
A part enters on its own position's entry overhang, so there is one destination a position. They are one vector differing in the 4 bases of that overhang; a block put in the wrong one cannot close.

Holes

2 numbers this protocol would otherwise have to invent. None is filled with a guess, and each says below what it waits on: a bench, a shelf, a price, or a number nobody has published.

H29 no sourced number — PCR1, the polymerase the cycle count is stated against: no cycle count published for Q5 on a Twist oligo pool. Waiting on a number nobody has published. Filled by a count Twist states against Q5, or a pilot on this pool.
H30 no sourced number — PCR2, the cycle count: no cycle count for PCR2, which pulls one block out of an already-amplified batch. Waiting on a number nobody has published. Filled by a pilot titrated against the heteroduplex hump on capillary electrophoresis, or a real-time run stopped before the curve plateaus.

Sources

Long, Y. et al. 2025, LevSeq, Supporting Information · held under reference_docs/ · 2026-10-06 https://doi.org/10.1021/acssynbio.4c00625 cited at step 1
Endura Competent Cells manual, Lucigen / Biosearch Technologies · MA133 26Feb2018 · plain curl · 2026-10-06 cited at p. 4-5
Twist Bioscience, Amplifying Twist Oligo Pools · REV 8 · plain curl · 2026-10-07 https://www.twistbioscience.com/content/dam/twistbioscience/resources/2026-01/FRM-001034-AmplifyingOligoPools-REV8%20singles.pdf cited at p. 2, cycle chart
liulab-mbio, Figure sources for four designs · docs/research/figure-sources.md cited at section 1, Baker's three-primer PCR design · section 2, Cargo GGA ligation
Twist Bioscience, Twist Oligo Pools Amplification Protocol · DOC-4060 REV 1.0 · held under reference_docs/ · docs/research/oligo-pool-pcr-cycles.md cited at Appendix B
New England Biolabs NEBioCalculator · script main-6f3020c533.js, capture 2026-08-28 · Wayback Machine · docs/research/primer-design-and-pcr.md cited at dsDNA molecular weight
New England Biolabs #E1602S/L NEBridge Golden Gate Assembly Kit (BsmBI-v2) instruction manual · version 3.0_6/26 · 2026-09-12 · docs/research/golden-gate-assembly.md https://www.neb.com/-/media/nebus/files/manuals/manuale1602.pdf cited at assembly protocol