Gibson assembly: GFP into pUC19

Open pUC19 by PCR across 395-452, amplify GFP with the overlap at each junction carried as a primer tail, join the 2 fragments in one NEBuilder HiFi DNA Assembly Master Mix reaction, and confirm the clone by colony PCR and sequencing.

Vector: pUC19, 2686 bp
Insert: GFP, 717 bp
Fragments: 2 in one reaction
Overlaps: 16 bp and 16 bp
Assembly: NEBuilder HiFi DNA Assembly Master Mix, 50 °C for 15 min
Product: pUC19-GFP, 3346 bp
Selection: ampicillin or carbenicillin

Keep in mind

One reaction joins 2 fragments: pUC19 backbone (2629 bp) and GFP (717 bp).

pUC19 backbone and GFP share 16 bp at 380, taken from pUC19 backbone and carried by the other as a primer tail.

GFP and pUC19 backbone share 16 bp at 1113, taken from pUC19 backbone and carried by the other as a primer tail.

GFP reads on the opposite strand from lac promoter, 60 bp away, so that promoter does not transcribe it.

There is no ribosome binding site annotated ahead of GFP, so the clone is not expected to make its protein.

The insertion interrupts lacZα, so correct clones are white and empty vector is blue on X-gal and IPTG.

pUC19 backbonepass
GFPpass
junctionspass
overlap lengthpass
overlap tmpass
overlap gcnot judged
overlap hairpinpass
overlap repeatpass
overlap similaritynot judged
fragment countpass
assembly DNApass
primerswarn

overlap gc not judged: pUC19 backbone-GFP 50%, GFP-pUC19 backbone 50%; no source quantifies a GC band for an overlap, so nothing judges this

overlap similarity not judged: pUC19 backbone-GFP and GFP-pUC19 backbone share 3 bases, the most any two here do; NEB asks for a unique overlap at each junction but nobody quantifies how alike is too alike, so nothing judges this

primers warn: 9 designed, 3 with a warning, 0 failing: GC on 2, Tm on 1; not judged: full-primer Tm, 3' end stability

Materials

Name	Supplier	Catalogue	Storage	Per run	Note	Source
pUC19 plasmid			-20 °C		PCR template
GFP template			-20 °C		PCR template
Q5 DNA Polymerase and its reaction buffer	New England Biolabs		-20 °C
dNTP mix			-20 °C		10 mM of each base
DpnI	New England Biolabs		-20 °C	20 units per PCR	cuts the methylated plasmid template only
Monarch Spin PCR & DNA Cleanup Kit	New England Biolabs	T1130
NEBuilder HiFi DNA Assembly Master Mix	New England Biolabs	E2621	-20 °C	10 µL per reaction
NEB 5-alpha Competent E. coli	New England Biolabs	C2987	-80 °C	50 µL per transformation
SOC or NEB 10-beta/Stable Outgrowth Medium				950 µL per transformation
LB agar plates with ampicillin or carbenicillin, 80 µg/mL X-gal and 200 µM IPTG				one plate per transformation		^[1]
Plasmid miniprep kit					for the clones that go to sequencing
OneTaq Quick-Load 2X Master Mix with Standard Buffer	New England Biolabs	M0486	-20 °C	12.5 µL per reaction
Agarose and 1X TAE or TBE
NEB 1 kb Plus DNA Ladder	New England Biolabs	N3200				^[2]
NEB 100 bp DNA Ladder	New England Biolabs	N3231				^[3]

Equipment: Thermocycler with a heated lid, Agarose gel rig and power supply, Microcentrifuge, Spectrophotometer (NanoDrop) or fluorometer (Qubit), Heat block or water bath at 42 °C, Shaking incubator and a plate incubator at 37 °C

Oligos

Order sheet: primers.tsv

Name	Sequence (5'→3')	Length	Tm (°C)	For	Working stock	Checks
pUC19 backbone forward	`GGCGTAATCATGGTCATAGC`	20	63.1	Amplify pUC19 backbone	10 µM	pass
pUC19 backbone reverse	`ACTGGCCGTCGTTTTACA`	18	64.1	Amplify pUC19 backbone	10 µM	warn
GFP forward	`TAAAACGACGGCCAGTATGAGTAAAGGAGAAGAACTTTTCAC`	42	62.1	Amplify GFP	10 µM	warn
GFP reverse	`TGACCATGATTACGCCCTATTTGTATAGTTCATCCATGCCAT`	42	62.0	Amplify GFP	10 µM	warn
Colony PCR forward	`TGGGTAACGCCAGGGTTTTCC`	21	61.9	Screen colonies by PCR	10 µM	pass
Colony PCR reverse	`CGTATGTTGTGTGGAATTGTGAGCGGAT`	28	62.2	Screen colonies by PCR	10 µM	pass
Junction reverse	`CATCACCTTCACCCTCTCCACTGA`	24	62.0	Screen colonies by PCR	10 µM	pass
Sequencing forward	`GCGCAACTGTTGGGAA`	16	63.3	Confirm the clone by sequencing	10 µM	pass
Sequencing reverse	`CGCAACGCAATTAATGTGAG`	20	62.2	Confirm the clone by sequencing	10 µM	pass

Checks on 3 of 9 oligos

pUC19 backbone reverse warn: Tm 64.1 °C (band 60-64)
GFP forward warn: GC 35% (band 40-60)
GFP reverse warn: GC 35% (band 40-60)

Make the fragments

1Amplify pUC19 backbone

Caution: Keep the polymerase on ice.

Thaw the buffer, dNTPs and primers on ice, then vortex and spin them down.
Mix the master mix, dispense it into each tube, then add the pUC19 template.
Run the program below: 64.1 °C annealing and 60 s extension for a 2629 bp product.

pUC19 backbone PCR

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.

pUC19 backbone PCR · 47 min 30 s plus ramps

Step	Temperature	Time	Cycles
Initial denaturation	98 °C	30 s	1
Denature	98 °C	10 s	×30^[4]
Anneal	64.1 °C	20 s
Extend	72 °C	1 min
Final extension	72 °C	2 min	1
Hold	4 °C	∞	1

pUC19 backbone PCR

Expected result

One band at 2629 bp.

Troubleshooting

No band: Drop the annealing temperature by 3 °C and check the pUC19 template is there.^[4]
Several bands: Raise the annealing temperature, or gel-purify the band of the right size.^[4]

Notes

Its primers carry no tail: neither of its junctions is this fragment's to carry.
The annealing temperature above is read from the annealing regions alone; a tail pairs with nothing on the template in the first cycles.

2Amplify GFP

Caution: Keep the polymerase on ice.

Thaw the buffer, dNTPs and primers on ice, then vortex and spin them down.
Mix the master mix, dispense it into each tube, then add the GFP template.
Run the program below: 63 °C annealing and 20 s extension for a 749 bp product.

GFP PCR

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.

GFP PCR · 27 min 30 s plus ramps

Step	Temperature	Time	Cycles
Initial denaturation	98 °C	30 s	1
Denature	98 °C	10 s	×30^[4]
Anneal	63 °C	20 s
Extend	72 °C	20 s
Final extension	72 °C	2 min	1
Hold	4 °C	∞	1

GFP PCR

Expected result

One band at 749 bp.

Troubleshooting

No band: Drop the annealing temperature by 3 °C and check the GFP template is there.^[4]
Several bands: Raise the annealing temperature, or gel-purify the band of the right size.^[4]

Notes

Its 5' tails are 16 bp of pUC19 backbone on the forward primer and 16 bp of pUC19 backbone on the reverse primer, so the amplicon already spells what its neighbours spell where they meet.
The annealing temperature above is read from the annealing regions alone; a tail pairs with nothing on the template in the first cycles.

3Check the PCRs on a gel

Use a 1% agarose gel.
Load 5 µL of each reaction beside the ladder.
Run until the dye front is two thirds down the gel.

Expected result

pUC19 backbone: one band at 2629 bp.
GFP: one band at 749 bp.

PCR products

L NEB 1 kb Plus DNA Ladder (N3200)
1 pUC19 backbone: 2629 bp
2 GFP: 749 bp

Troubleshooting

A smear or an extra band: Gel-purify the band of the right size; a wrong template in the assembly gives wrong clones.^[5]

Notes

1% agarose resolves bands of these sizes.^[6]

4Digest the plasmid template with DpnI

Add 20 units of DpnI to the pUC19 backbone PCR and mix.
Incubate at 37 °C for 30 minutes.
Heat-inactivate DpnI at 80 °C for 20 minutes.

DpnI digestHeat-inactivate DpnI

Expected result

Nothing visible. The digest shows up later as fewer colonies carrying the template plasmid.

Troubleshooting

Many colonies on the no-insert control: The template survived: digest longer, or use more DpnI.^[5]

Notes

DpnI cuts GATC only where Dam has methylated it, so it cuts pUC19 (15 Dam sites) and leaves the PCR product, which carries no methylation.^[7]
The dose, the time and the heat inactivation are the assembly manual's, not this package's.^[8]
The other answer to template background is to keep it low in the first place: 0.1-0.5 ng of plasmid per 50 µL PCR.^[8]

5Purify every amplicon

Run each reaction over a Monarch Spin PCR & DNA Cleanup column and elute in the smallest volume the kit allows.

Expected result

Clean DNA, free of polymerase, primers and dNTPs.

Troubleshooting

Low recovery: Elute in a larger volume and leave the buffer on the column longer.^[9]

Notes

A column is optional below 3 PCR fragments: a product more than 90% pure goes into the reaction unpurified, within the fraction the assembly step gives.^[8]
At 3 fragments or more, or a fragment over 5 kb, a column is highly recommended, and the gain in assembly and transformation is put at two- to tenfold. A PCR showing anything but one band is gel-purified whatever the count.^[8]

6Measure every concentration

Measure each purified amplicon by A260 (NanoDrop) or with a fluorometer (Qubit).
Enter each concentration in the next table's Stock column.

Expected result

pUC19 backbone: 0.0309 pmol is 50.04 ng, so 50 ng/µL or more fits in 1 µL.
GFP: 0.0618 pmol is 27.29 ng, so 27 ng/µL or more fits in 1 µL.

Troubleshooting

Too dilute to fit in the reaction: Concentrate the amplicon, or scale the reaction up.

Assemble

7Set up the NEBuilder HiFi DNA Assembly Master Mix reaction

Thaw the master mix on ice and mix it well.
Pipette the DNA into the tube first, then the master mix.
Mix gently and spin down.

NEBuilder HiFi DNA Assembly Master Mix reaction

Reactions mix includes 10% extra

Component	Stock	Final	1 rxn (µL)	Mix for 1 (µL)
pUC19 backbone^[10]	ng/µL	0.0309 pmol (50.04 ng)	1	each tube
GFP^[10]	ng/µL	0.0618 pmol (27.29 ng)	1	each tube
NEBuilder HiFi DNA Assembly Master Mix^[8]	2X	1X	10	11
Nuclease-free water		to 20 µL	8	8.8
Total			20	19.8 mix only

Put 18 µL of mix in each tube, then add 1 µL pUC19 backbone, 1 µL GFP.

Expected result

A 20 µL reaction holding every fragment.

Troubleshooting

The DNA does not fit the reaction volume: Concentrate the fragments, or scale the whole reaction up and add master mix in proportion.^[8]
Colonies later carry the empty vector: The backbone PCR carried its plasmid template through; digest it with DpnI again, or gel-purify the backbone.^[8]

Notes

Each insert goes in at 2 times the vector's moles, and any insert under 200 bp at 5 times, at this fragment count. The table gives each in picomoles and in nanograms, so it can be pipetted at whatever concentration it is.^[8]
Unpurified PCR product goes straight from the tube for up to 20% of the reaction, which is 4 µL here, as long as the product is a single band.^[8]

8Incubate the assembly

Put the tube in the thermocycler and run the program below.
Put the reaction on ice afterwards, or keep it at -20 °C.

Assembly incubation · 15 min plus ramps

Step	Temperature	Time	Cycles
Assembly	50 °C	15 min	1^[8]
Hold	4 °C	∞	1

Assembly incubation

pUC19-GFP as the incubation closes it: the overlaps the parts share, 16 bp and 16 bp.

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Expected result

Nothing visible. One exonuclease, one polymerase and one ligase work together at this one temperature; there is nothing to cycle.
The product spells TAAAACGACGGCCAGT at 380, where pUC19 backbone meets GFP.
The product spells GGCGTAATCATGGTCA at 1113, where GFP meets pUC19 backbone.

Troubleshooting

Few colonies later: Run the kit's positive control beside the assembly; it tells a bad master mix from a bad design.^[8]
No colonies later: Run the reaction on a gel: an efficient assembly shows the fragments gone and a product of the right size.^[8]

Notes

15 minutes is what this fragment count takes. Incubating longer than 15 minutes improves some assemblies, up to four hours; overnight is refused.^[8]
Junction positions are 1-based, on the product.

Transform and screen

9Transform and plate

Caution: Competent cells die if they warm up; keep them on ice until the shock.

Thaw 50 µL of NEB 5-alpha Competent E. coli (C2987) on ice for 10 minutes.
Add 2 µL of the assembly and flick the tube four or five times.
Hold on ice for 30 minutes.
Heat shock at 42 °C for 30 seconds.
Return to ice for 5 minutes.
Add 950 µL of outgrowth medium and shake at 37 °C for 60 minutes at 250 rpm.
Spread 50 µL of a 1:5 dilution on a warmed plate and grow overnight at 37 °C.

ThawOn iceHeat shockBack on iceOutgrowth

Expected result

Hundreds of colonies. NEB's own lot test asks for more than 100 from a six-fragment assembly with a tenth of the outgrowth plated; this reaction joins 2.
Correct clones are white and empty vector is blue: the insertion interrupts lacZα, which is then not there to complete the host's own.

Troubleshooting

No colonies: Check the antibiotic and the cells' efficiency, and plate the rest of the outgrowth.^[5]
A lawn: Plate a smaller volume or a greater dilution next time.

Notes

The plate reads colour only with an alpha-complementing host, such as NEB 5-alpha Competent E. coli (C2987). A host that cannot complement gives white colonies whatever the clone carries.
The clone is not expected to make GFP: it reads on the opposite strand from lac promoter and no ribosome binding site is annotated ahead of it.

10Screen colonies by PCR

Touch a well-separated colony with a sterile toothpick and stir it into the tube until the liquid clouds.
Streak the same toothpick onto a numbered plate, so the clone survives the PCR.
Run the program below and load 5 µL on a 2% gel.

Colony PCR

Reactions mix includes 10% extra

Component	Stock	Final	1 rxn (µL)	Mix for 1 (µL)
OneTaq Quick-Load 2X Master Mix with Standard Buffer (M0486)	2X	1X	12.5	13.8
Forward primer	10 µM	200 nM	0.5	0.55
Reverse primer	10 µM	200 nM	0.5	0.55
Nuclease-free water		to 25 µL	11.5	12.7
Total			25	27.5

Put 25 µL of mix in each tube.

Colony PCR · 1 h 10 min plus ramps

Step	Temperature	Time	Cycles
Lysis	94 °C	5 min	1
Denature	94 °C	30 s	×30^[11]
Anneal	56.9 °C	30 s
Extend	68 °C	1 min
Final extension	68 °C	5 min	1
Hold	10 °C	∞	1

Colony PCR

Expected result

Each of the 2 junctions is read: the flanking pair crosses them all, and each junction primer stops inside its own insert.
Correct clone: 160 bp, 837 bp.
Empty vector: 177 bp.

Colony PCR

L NEB 100 bp DNA Ladder (N3231)
1 Correct clone: 160, 837 bp
2 Empty vector: 177 bp

Troubleshooting

No band in any lane: The colony was too much material: touch a smaller one, or dilute it.^[12]
Nothing grew at all: Run the same PCR on the assembly reaction itself, with primers flanking the assembled product.^[8]
Every colony reads as empty vector: A PCR-generated vector carries uncut plasmid through; digest it with DpnI again, or gel-purify the backbone.^[8]
A colony gives a band of the wrong size: The PCR that made a part was not a single band; gel-purify it and assemble again. NEB Stable Competent E. coli (#C3040) suits an insert carrying repeats.^[8]

Notes

The long first step at 94 °C lyses the cells; there is no purified template.
This screen reads 10 colonies.
The correct fraction falls with fragment count: 10 of 10 correct at two fragments, 4 of 10 at five. That is the In-Fusion series' number, the only one measured against fragment count, and not this product's.^[13]

11Confirm the clone by sequencing

Miniprep two or three colonies that read as correct.
Send each miniprep with both sequencing primers.
Check each read against product.dna; keep a clone only when every junction and insert reads true.

Expected result

Sequencing forward anneals 123 bp from its own junction and has to read 840 bp to cover the far one.
Sequencing reverse anneals 118 bp from its own junction and has to read 835 bp to cover the far one.
The junctions read as TAAAACGACGGCCAGT and GGCGTAATCATGGTCA, and the parts match GFP.

Troubleshooting

The read starts too close to the junction: Move the primer further out; the first bases after a primer are unreadable.^[14]

Notes

Only a read across the junctions confirms the assembly, whatever the screen said.
A provider whose read is shorter than the lengths above needs a further primer inside the inserts.
About one error per 50 molecules joined was found in 210 sequenced junctions, so a gel that reads right is not the same as a junction that is right.^[15]

Sources

Potapov, V. et al. (2018) Comprehensive profiling of four base overhang ligation fidelity by T4 DNA Ligase and application to DNA assembly. ACS Synth. Biol. 7, 2665-2674 · docs/research/golden-gate-assembly.md https://doi.org/10.1021/acssynbio.8b00333 cited at methods
New England Biolabs #N3200 1 kb Plus DNA Ladder product page · capture 2026-03-07 · Wayback Machine · docs/research/primer-design-and-pcr.md cited at band sizes
New England Biolabs #N3231 100 bp DNA Ladder product page · capture 2025-10-31 · Wayback Machine · docs/research/primer-design-and-pcr.md cited at band sizes
New England Biolabs #M0491 Q5 High-Fidelity DNA Polymerase protocol page · capture 2024-08-04 · Wayback Machine · 2026-09-12 cited at thermocycling conditions · annealing temperature
New England Biolabs, Troubleshooting Guide for Cloning · 2026-09-18 · docs/research/restriction-ligation.md https://www.neb.com/en-us/tools-and-resources/troubleshooting-guides/troubleshooting-guide-for-cloning cited at colonies contain the wrong construct · few or no transformants
New England Biolabs, Agarose Gel Resolution · capture 2024-09-18 · Wayback Machine · docs/research/primer-design-and-pcr.md cited at optimum resolution for linear DNA
REBASE enzyme record for DpnI · docs/research/golden-gate-assembly.md http://rebase.neb.com/rebase/rebase.html cited at DpnI
New England Biolabs #E2621S/L/X and #E5520S NEBuilder HiFi DNA Assembly Master Mix / Cloning Kit instruction manual · version 6.0_1/26 https://www.neb.com/-/media/nebus/files/manuals/manuale2621_e5520.pdf cited at reaction setup · reaction setup, scale footnote · troubleshooting, clones without the insert · incubation · troubleshooting, no colonies · troubleshooting, wrong product
New England Biolabs #T1130 Monarch Spin PCR & DNA Cleanup Kit instruction manual · version 1.0 06.24 · 2026-09-18 · docs/research/restriction-ligation.md cited at troubleshooting, low DNA yield
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 #M0480 OneTaq DNA Polymerase protocol · protocols.io version 2, 2022-02-21 · 2026-09-12 https://dx.doi.org/10.17504/protocols.io.bd24i8gw cited at thermocycling conditions
New England Biolabs application note, Robust Colony PCR from Multiple E. coli Strains using OneTaq Quick-Load Master Mixes · Y. Xu, 11/13 · 2026-09-12 · docs/research/primer-design-and-pcr.md cited at template
Takara Bio, In-Fusion Cloning FAQs · 2026-09-18 https://www.takarabio.com/learning-centers/cloning/in-fusion-cloning-faqs
Azenta/Genewiz, Sanger sequencing FAQ and technical notes · 2026-09-12 · docs/research/primer-design-and-pcr.md cited at sequencing primer design
Gibson, D.G. et al. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343-345 https://doi.org/10.1038/nmeth.1318