Reagents and equipment

Everything the whole run takes, each row naming the protocols that take it. Each protocol page carries its own list, which is what to lay out before that sitting.

Materials

Name	Supplier	Catalogue	Storage	Per run	Source	Used in
pCR-Blunt II-TOPO			-20 °C			Part carrier
Zero Blunt TOPO PCR Cloning Kit			-20 °C	one reaction per well	^[2]	Part carrier
nuclease-free water				25,160 µL in all		Primer plates
Empty 96-well plate				1 for the working plates		Primer plates
Adhesive plate seals				2 seals		Primer plates
Oligo pool			-20 °C	131 oligos, every one 350 nt, resuspended to 20 ng/µL		Cargo ordering and pool preparation
NEBridge Golden Gate Assembly Kit, BsmBI-v2	NEB	E1602	-20 °C	one assembly per block		Cargo ordering and pool preparation, Cargo creation
Q5 DNA Polymerase	NEB	M0491	-20 °C	one reaction per batch, then one per block		Cargo creation
Pool amplification primers			-20 °C	74 primers: 1 P1 batch forward, 72 P2 gene reverse, 1 P3 batch outer		Cargo creation
dNTP mix	NEB	N0447	-20 °C	0.2 µL a reaction, 10 mM each	^[7]	Cargo creation, Cargo validation: index PCR
DMX-iGGA vector			-20 °C	3, one a position		Cargo creation, Library assembly in rounds
BbsI-HF	New England Biolabs	R3539	-20 °C	2.5 µL per digest		Cargo creation, Library assembly in rounds
SrfI	New England Biolabs	R0629	-20 °C	2.5 µL per digest		Cargo creation, Library assembly in rounds
CutSmart Buffer			-20 °C			Cargo creation, Library assembly in rounds, Final cargo ligation
SPRI paramagnetic beads				2 volumes after a digest, 1 after a ligation		Cargo creation, Library assembly in rounds, Final cargo ligation
Endura ElectroCompetent Cells	Lucigen	60242-2	-80 °C	4 aliquots, one a pulse, 25 µL each	^[4]	Cargo creation, Library assembly in rounds, Final cargo ligation
Recovery medium				one outgrowth per round		Cargo creation, Library assembly in rounds
Selective broth and plates						Cargo creation, Library assembly in rounds
25 cm BioAssay plate	Corning	431111		one spot a design	^[10]	Cargo validation: barcode ligation, Cargo validation: index PCR
384-well culture plate	Beckman Coulter	c74290		1, one a 384 wells	^[10]	Cargo validation: barcode ligation, Cargo validation: index PCR
1536-well barcoding plate	Greiner	782270		1, one a 4 picked plates	^[10]	Cargo validation: barcode ligation
DMX barcode kit			-20 °C	96 plasmids, 4 groups of 24		Cargo validation: barcode ligation
Barcoding master mix			-20 °C	0.5 µL a well	^[10]	Cargo validation: barcode ligation
96-well half-skirted PCR plate	USA Scientific	1402-9700		3, one a quarter of a picked plate	^[7]	Cargo validation: index PCR
Barcoded index primer plate			-20 °C	2 µL a well at 1 µM each, 96 forward and 96 reverse	^[7]	Cargo validation: index PCR
ThermoPol Reaction Buffer	NEB	B9004	-20 °C	1 µL a reaction, 10X	^[7]	Cargo validation: index PCR
Taq DNA Polymerase	NEB	M0267	-20 °C	0.05 µL a reaction	^[12]	Cargo validation: index PCR
DMSO, molecular biology grade	MP Biomedicals	194819		0.4 µL a reaction, 4% (v/v) in the well	^[7]	Cargo validation: index PCR
N part list			-20 °C	24 members, pooled		Library assembly in rounds
DBD part list			-20 °C	24 members, pooled		Library assembly in rounds
C part list			-20 °C	24 members, pooled		Library assembly in rounds
BsaI-HFv2	New England Biolabs	R3733	-20 °C	2.5 µL per digest		Library assembly in rounds, Final cargo ligation
PmeI	New England Biolabs	R0560	-20 °C	2.5 µL per digest		Library assembly in rounds, Final cargo ligation
T7 DNA Ligase	NEB	#M0318L	-20 °C		^[13]	Library assembly in rounds, Final cargo ligation
StickTogether DNA Ligase Buffer	NEB	#B0535S	-20 °C		^[13]	Library assembly in rounds, Final cargo ligation
Plasmid prep kit				one prep per round		Library assembly in rounds, Final cargo ligation
Electroporation cuvettes				4, one a pulse		Library assembly in rounds, Final cargo ligation
pLVX-TetOne-dom vector			-20 °C			Final cargo ligation
PaqCI	New England Biolabs	R0745	-20 °C	2.5 µL per digest		Final cargo ligation
Selective plates for the final transfer						Final cargo ligation

T7 DNA Ligase: never PEG Never add PEG to a T7 ligase reaction. Blunt-end ligation is forced above 20% (w/v) PEG 6000, and StickTogether buffer already carries 7.5%; the refusal that removes escapees holds only while nothing adds more.^[13]
T7 DNA Ligase: never heat inactivation Do not heat-inactivate T7 DNA ligase in PEG: transformation will be inhibited. Go from the ligation straight to the clean-up.^[13]

Caution: Keep the polymerase on ice.

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

Equipment and plasticware

Part carrier plate 96 wells used in Part carrier
Multichannel pipette used in Primer plates
Plate shaker used in Primer plates
Plate centrifuge used in Primer plates
primer stock plate 96 wells used in Primer plates
primer working plate 1 96 wells used in Primer plates
Thermocycler taking a 96-well plate used in Cargo creation, Cargo validation: index PCR
Gel tank and a transilluminator used in Cargo creation
PCR2 plate 96 wells used in Cargo creation
QPix XE Microbial Colony Picker used in Cargo validation: barcode ligation, Cargo validation: index PCR
ECHO 525 acoustic liquid handler used in Cargo validation: barcode ligation
Incubator at 37 °C used in Cargo validation: barcode ligation, Cargo validation: index PCR
A sequencer, and a demultiplexer that can check an address used in Cargo validation: barcode ligation, Cargo validation: index PCR
picked 1 384 wells · Beckman Coulter #c74290 used in Cargo validation: barcode ligation, Cargo validation: index PCR
lysate 1 1536 wells · Greiner #782270 used in Cargo validation: barcode ligation
multichannel pipette on 96-well spacing used in Cargo validation: index PCR
index 1 96 wells · USA Scientific #1402-9700 used in Cargo validation: index PCR
index 2 96 wells · USA Scientific #1402-9700 used in Cargo validation: index PCR
index 3 96 wells · USA Scientific #1402-9700 used in Cargo validation: index PCR
Incubator or heat block at 37 °C used in Library assembly in rounds, Final cargo ligation
Magnetic rack for the bead clean-ups used in Library assembly in rounds, Final cargo ligation
Electroporator and cuvettes used in Library assembly in rounds, Final cargo ligation
Shaking incubator at 30 °C used in Library assembly in rounds, Final cargo ligation
Spectrophotometer (NanoDrop) or fluorometer (Qubit) used in Library assembly in rounds, Final cargo ligation
Long-read sequencer, for the linkage read of the finished library used in Library assembly in rounds
Short-read sequencer, for the representation read of the finished library used in Library assembly in rounds
Short-read sequencer, for the final representation read of the finished library used in Final cargo ligation

What this run consumes

Every item this run consumes has a row. A quantity comes from the design and is here whatever is loaded; one the design does not size carries what the protocol states. A charge comes only from a price record; where none prices a row, the money is a hole and no figure is estimated. Price steers no part of the design.

Item	Key	Quantity	Charge (USD)	Headroom
AP-1 DESynR oligo pool	oligo-pool	131 oligos	2575.00^[1]	131 count, 369 below the next band; 350 length, no slack above it at all
Pool amplification primers	pool-primers	74 primers	370.00^[1]	74 count, 22 below the next band
BbsI-HF	R3539	7.5 µL	41.50^[1]
BsaI-HFv2	R3733	7.5 µL	12.45^[1]
SrfI	R0629	7.5 µL	27.90^[1]
PmeI	R0560	7.5 µL	13.20^[1]
Endura ElectroCompetent Cells	60242-2	3 aliquots	71.04^[1]	3 count, 21 below the next band
Electroporation cuvettes	cuvettes	3 cuvettes	11.82^[1]	3 count, 47 below the next band
Plasmid prep kit	plasmid prep	3 preps	7.68^[1]	3 count, 47 below the next band
pCR-Blunt II-TOPO	pCR-Blunt II-TOPO	no sourced number	no sourced number
Zero Blunt TOPO PCR Cloning Kit	Zero Blunt TOPO PCR Cloning Kit	one reaction per well	no sourced number
nuclease-free water	nuclease-free water	25,160 µL in all	no sourced number
Empty 96-well plate	Empty 96-well plate	1 for the working plates	no sourced number
Adhesive plate seals	Adhesive plate seals	2 seals	no sourced number
NEBridge Golden Gate Assembly Kit, BsmBI-v2	E1602	one assembly per block	no sourced number
Q5 DNA Polymerase	M0491	one reaction per batch, then one per block	no sourced number
dNTP mix	N0447	0.2 µL a reaction, 10 mM each	no sourced number
DMX-iGGA vector	DMX-iGGA vector	3, one a position	no sourced number
CutSmart Buffer	CutSmart Buffer	no sourced number	no sourced number
SPRI paramagnetic beads	SPRI paramagnetic beads	2 volumes after a digest, 1 after a ligation	no sourced number
Recovery medium	Recovery medium	one outgrowth per round	no sourced number
Selective broth and plates	Selective broth and plates	no sourced number	no sourced number
25 cm BioAssay plate	431111	one spot a design	no sourced number
384-well culture plate	c74290	1, one a 384 wells	no sourced number
1536-well barcoding plate	782270	1, one a 4 picked plates	no sourced number
DMX barcode kit	DMX barcode kit	96 plasmids, 4 groups of 24	no sourced number
Barcoding master mix	Barcoding master mix	0.5 µL a well	no sourced number
96-well half-skirted PCR plate	1402-9700	3, one a quarter of a picked plate	no sourced number
Barcoded index primer plate	Barcoded index primer plate	2 µL a well at 1 µM each, 96 forward and 96 reverse	no sourced number
ThermoPol Reaction Buffer	B9004	1 µL a reaction, 10X	no sourced number
Taq DNA Polymerase	M0267	0.05 µL a reaction	no sourced number
DMSO, molecular biology grade	194819	0.4 µL a reaction, 4% (v/v) in the well	no sourced number
N part list	N part list	24 members, pooled	no sourced number
DBD part list	DBD part list	24 members, pooled	no sourced number
C part list	C part list	24 members, pooled	no sourced number
T7 DNA Ligase	#M0318L	no sourced number	no sourced number
StickTogether DNA Ligase Buffer	#B0535S	no sourced number	no sourced number
pLVX-TetOne-dom vector	pLVX-TetOne-dom vector	no sourced number	no sourced number
PaqCI	R0745	2.5 µL per digest	no sourced number
Selective plates for the final transfer	Selective plates for the final transfer	no sourced number	no sourced number
Total			3130.59