DNA MODIFYING ENZYMES

Gibson Assembly ™ Master Mix

Instruction Manual

NEB #E2611S/L
10/50 reactions
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GIBSON ASSEMBLY™ is a trademark of Synthetic Genomics, Inc.
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Gibson Assembly Master Mix

Table of Contents:
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Specification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Overview of Gibson Assembly Master Mix Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Design and PCR Amplification of Fragments for Gibson Assembly . . . . . . . . . . . . . 3
Gibson Assembly Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Chemically Competent Cells Transformation Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Electrocompetent Cells Transformation Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Usage Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Frequently Asked Questions (FAQs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Components:
Store at –20°C. Thaw, vortex thoroughly before use and keep on ice.

Gibson Assembly Master Mix (2X)

Positive Control
2 overlapping dsDNA fragments for control assembly.

Required Materials Not Included:

DNA Polymerases (for generating PCR products):
We recommend Q5™ High-Fidelity DNA Polymerase (NEB #M0491) or related products,
such as Q5 Hot Start Flex DNA Polymerase (NEB #M0493), Q5 Hot Start Flex 2X Master
Mix (NEB #M0494).

LB (Luria-Bertani) plates with appropriate antibiotic.

SOC Outgrowth Medium (NEB #B9020).
Competent Cells:
We recommend NEB 5-alpha Competent E. coli (High Efficiency, NEB #C2987).
For assembled products greater than 10 kb, NEB recommends using NEB 10-beta
Competent E. coli (High Efficiency, NEB #C3019) or NEB 10-beta Electrocompetent E. coli
(NEB #C3020).

1
 Introduction:
Gibson Assembly was developed by Dr. Daniel Gibson and his colleagues at
the J. Craig Venter Institute and licensed to NEB by Synthetic Genomics, Inc. It
allows for successful assembly of multiple DNA fragments, regardless of frag-
ment length or end compatibility. It has been rapidly adopted by the synthetic
biology community due to its ease-of-use, flexibility and suitability for large DNA
constructs.
Gibson Assembly efficiently joins multiple overlapping DNA fragments in a
single-tube isothermal reaction (1,2). The Gibson Assembly Master Mix includes
three different enzymatic activities that perform in a single buffer:
• The exonuclease creates single-stranded 3´ overhangs that facilitate the
annealing of fragments that share complementarity at one end (overlap region).
• The proprietary polymerase fills in gaps within each annealed fragment.
• The DNA ligase seals nicks in the assembled DNA.
The end result is a double-stranded fully sealed DNA molecule that can serve
as template for PCR, RCA or a variety of other molecular biology applications,
including direct transformation. The method has been successfully used by Gib-
son’s group and others to assemble oligonucleotides, DNA with varied overlaps
(15–80 bp) and fragments hundreds of kilobases long (1–2).

Figure 1: Overview of the Gibson Assembly Method

dsDNA fragments with overlapping ends.

A
3´
5´ B
5´
3´

Gibson Assembly

Add fragments to 3´
Gibson Assembly 5´
5´
Master Mix. 3´
5´ Exonuclease chews back 5´ ends.
3´
5´
5´
3´

DNA fragments anneal.

3´ 5´
5´ 3´

DNA polymerase extends 3´ ends.

Incubate at 50°C
for 1 hour.
DNA ligase seals nicks.

A+B
Fully Assembled DNA

2
Specification:
10 µl of 2X Gibson Assembly Master Mix was incubated with 6 fragments
(5 fragments of 400 bp and one of 2,780 bp, with 40 bp overlap, 0.05 pmol
each) in a final volume of 20 µl at 50°C for 60 minutes. NEB 5-alpha Competent
E. coli (NEB #C2987) were transformed with 2 µl of the master mix/fragment
mixture using the transformation protocol on page 11. Greater than 100
white colonies were observed when 1/10 of the outgrowth was spread on an
ampicillin plate with IPTG/Xgal and incubated overnight.

Overview of Gibson Assembly Master Mix Protocol:

• Design primers to amplify fragments (and/or vector) with appropriate
overlaps (see pages 3–7)
• PCR amplify fragments and/or vector using a high fidelity DNA polymerase
(DNA can also be prepared using a restriction digest)
• Confirm and determine concentration of fragments using agarose gel
electrophoresis, a Nanodrop™ instrument or other method
• Add DNAs to Gibson Assembly Master Mix and incubate at 50°C for 1 hour
• Transform into E. coli or use directly in other applications

Design and PCR Amplification of Fragments for

Gibson Assembly:
Note: We highly recommend using our web tool, NEBuilder™, available at www.
NEBGibson.com, to design PCR primers with overlapping sequences between
the adjacent DNA fragments and for their assembly into a cloning vector.

NEBuilder is the fastest and easiest approach to obtain ready-to-use

sequences for overlapping primers. However, it does not give details about
the primer design workflow. In some cases, it might be appropriate to further
manually alter primer sequences in order to adapt them for the use in more
complex assemblies, such as those that include site-specific mutagenesis.
For this purpose, it is absolutely necessary to understand the general
requirements and rules that apply to PCR primers used in conjunction with
Gibson Assembly. The sections below offer step-by-step directions and
recommendations for the manual design of primers for the assembly of two or
more PCR fragments, as well as primer design for assembly of PCR fragments
into a cloning vector prepared either by PCR or by restriction digestion.
3
 Structure of the Overlapping Primers
PCR primers for use in Gibson Assembly must have two sequence
components:
• an overlap sequence, required for the assembly of adjacent fragments;
• a gene-specific sequence, required for template priming during PCR
amplification;
The non-priming overlap sequence is added at the 5´-end of the primer.
This sequence is homologous to the 5´-terminal sequence of the adjacent
fragment. The length of overlap sequence is dependent on the GC content of
the sequences.
The priming gene-specific sequence is added at the 3´-end of the primer after
the overlap sequence. The priming sequence should meet the criteria required
for template annealing during PCR amplification.
The Tm of the 3´ gene-specific sequence of the primer can be
calculated using the Tm calculator found on the NEB website at http://
www.neb.com/TmCalculator.

General Recommendations for Design of Overlapping Primers

To achieve efficient assembly of PCR fragments into a vector, we suggest
using a 15–25 nt overlap with a Tm equal to or greater than 48°C (assuming
A-T pair = 2°C and G-C pair = 4°C, Fig. 2A, Step I). To prevent errors in primer
design it is highly recommended to first perform DNA fragment assembly in
silico and create a final sequence file displaying both DNA strands. This virtual
sequence may then be used as a template to design overlapping primers.
Figure 2A shows the workflow for overlapping primer design by using an
in silico-created DNA sequence file. First, mark the junctions between the
adjacent fragments 1, 2 and 3 (Fig. 2A, step II). Next, at or near each junction
choose 15–25 nucleotide sequences to serve as the overlap region between
the two adjacent fragments (Fig. 2A, step III). For the best fit, in terms of
length and Tm, the overlap sequence can be composed of nucleotides which
belong to only one fragment (overlap shown in blue) or it can be split between
the two adjacent fragments in any combination (overlap shown in orange).
Mark the first 5´ and the last 3´ nucleotide of the overlap sequence on both
DNA strands (boxed sequence). Finally, starting from the first 5´ nucleotide,
copy the entire overlap sequence in the 5´ to 3´ direction and, if necessary,
continue to add additional nucleotides to the 3´ end until the gene-specific
priming sequence length is reached (Fig. 2A, step IV). The reverse overlapping
primer is designed following the same steps as described above but copying
the sequence from the complementary DNA strand in the 5´ to 3´ direction.
Keep in mind that the two primers sharing the same overlap sequence are
always used in separate PCR reactions, each in combination with the primer,
which primes the complementary sequence on the opposite end of the
respective DNA fragment (Fig. 2A, step V).
4
Figure 2A: Primer design using in silico created final DNA sequence file.
I. Create a final sequence file displaying both DNA strands:
Fragment 1 Fragment 2 Fragment 3

ATGACCATGATTACGGATTC...TGAGCGGCATTTTCCGTGAC GTCTCGTTGCTGCATAAACC...GCGAGGTGCGGATTGAAAAT GGTCTGCTGCTGCTGAACGG...AGTTGGTCTGGTGTCAAAAA

TACTGGTACTAATGCCTAAG...ACTCGCCGTAAAAGGCACTG CAGAGCAACGACGTATTTGG...CGCTCCACGCCTAACTTTTA CCAGACGACGACGACTTGCC...TCAACCAGACCAGAGTTTTT

II. Mark adjacent fragment junctions:

ATGACCATGATTACGGATTC...TGAGCGGCATTTTCCGTGAC GTCTCGTTGCTGCATAAACC...GCGAGGTGCGGATTGAAAAT GGTCTGCTGCTGCTGAACGG...AGTTGGTCTGGTGTCAAAAA
TACTGGTACTAATGCCTAAG...ACTCGCCGTAAAAGGCACTG CAGAGCAACGACGTATTTGG...CGCTCCACGCCTAACTTTTA CCAGACGACGACGACTTGCC...TCAACCAGACCAGAGTTTTT

III. Choose 15-25 nt overlap region between two adjacent fragments:

ATGACCATGATTACGGATTC...TGAGCGGCATTTTCCGTGAC GTCTCGTTGCTGCATAAACC...GCGAGGTGCGGATTGAAAAT GGTCTGCTGCTGCTGAACGG...AGTTGGTCTGGTGTCAAAAA
TACTGGTACTAATGCCTAAG...ACTCGCCGTAAAAGGCACTG CAGAGCAACGACGTATTTGG...CGCTCCACGCCTAACTTTTA CCAGACGACGACGACTTGCC...TCAACCAGACCAGAGTTTTT

IV. Design overlapping primers starting from the first 5´ nucleotide of the overlap region:
Primer FP1 Primer FP2 Primer FP3

ATGACCATGATTACGGATTC...TGAGCGGCATTTTCCGTGAC GTCTCGTTGCTGCATAAACC...GCGAGGTGCGGATTGAAAAT GGTCTGCTGCTGCTGAACGG...AGTTGGTCTGGTGTCAAAAA

TACTGGTACTAATGCCTAAG...ACTCGCCGTAAAAGGCACTG CAGAGCAACGACGTATTTGG...CGCTCCACGCCTAACTTTTA CCAGACGACGACGACTTGCC...TCAACCAGACCAGAGTTTTT

Primer RP1 Primer RP2 Primer RP3

V. Amplify Fragment 1 with primers FP1 + RP1, Fragment 2 with primers FP2 + RP2 and Fragment 3 with primers FP3 + RP3:

Fragment 1 Overlap Overlap Fragment 3

ATGACCATGATTACGGATTC...TGAGCGGCATTTTCCGTGACGTCTCGTTGC GGTCTGCTGCTGCTGAACGG...AGTTGGTCTGGTGTCAAAAA
TACTGGTACTAATGCCTAAG...ACTCGCCGTAAAAGGCACTGCAGAGCAACG CCAGACGACGACGACTTGCC...TCAACCAGACCAGAGTTTTT
Fragment 2
GTGACGTCTCGTTGCTGCATAAACC...GCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTG
CACTGCAGAGCAACGACGTATTTGG...CGCTCCACGCCTAACTTTTACCAGACGACGACGAC

Figure 2B: Primer design for PCR-generated vector and insert using in silico
created final DNA sequence file.
I. Create a final sequence file displaying both DNA strands:
Vector Left Arm Insert Vector Right Arm

GTTTAACTTTAAGAAGGAGATATACAT ATGACCATGATTACGGATTCACT...AGTTGGTCTGGTGTCAAAAATAA TGAGATCCGGCTGCTAACAAAGCCCGAAA

CAAATTGAAATTCTTCCTCTATATGTA TACTGGTACTAATGCCTAAGTGA...TCAACCAGACCAGAGTTTTTATT ACTCTAGGCCGACGATTGTTTCGGGCTTT

II. Mark the junctions between the vector and the insert:
GTTTAACTTTAAGAAGGAGATATACAT ATGACCATGATTACGGATTCACT...AGTTGGTCTGGTGTCAAAAATAA TGAGATCCGGCTGCTAACAAAGCCCGAAA
CAAATTGAAATTCTTCCTCTATATGTA TACTGGTACTAATGCCTAAGTGA...TCAACCAGACCAGAGTTTTTATT ACTCTAGGCCGACGATTGTTTCGGGCTTT

III. Choose 15-25 nt overlap region between vector and insert:

GTTTAACTTTAAGAAGGAGATATACAT ATGACCATGATTACGGATTCACT...AGTTGGTCTGGTGTCAAAAATAA TGAGATCCGGCTGCTAACAAAGCCCGAAA
CAAATTGAAATTCTTCCTCTATATGTA TACTGGTACTAATGCCTAAGTGA...TCAACCAGACCAGAGTTTTTATT ACTCTAGGCCGACGATTGTTTCGGGCTTT

IV. Design overlapping primers starting from the first 5´ nucleotide of the overlap region:
Primer FP1 Primer FP2

GTTTAACTTTAAGAAGGAGATATACAT ATGACCATGATTACGGATTCACT...AGTTGGTCTGGTGTCAAAAATAA TGAGATCCGGCTGCTAACAAAGCCCGAAA

CAAATTGAAATTCTTCCTCTATATGTA TACTGGTACTAATGCCTAAGTGA...TCAACCAGACCAGAGTTTTTATT ACTCTAGGCCGACGATTGTTTCGGGCTTT

Primer RP2 Primer RP1

V. Amplify Insert with primers FP1 + RP1 and Vector with primers FP2 + RP2:
Vector Left Arm Vector Right Arm

Overlap Overlap
GTTTAACTTTAAGAAGGAGATATACAT ATGACC TGAGATCCGGCTGCTAACAAAGCCCGAAA
CAAATTGAAATTCTTCCTCTATATGTA TACTGG ACTCTAGGCCGACGATTGTTTCGGGCTTT
GGAGATATACAT ATGACCATGATTACGGATTCACT...AGTTGGTCTGGTGTCAAAAATAA TGAGATCCGGCTGCT
CCTCTATATGTA TACTGGTACTAATGCCTAAGTGA...TCAACCAGACCAGAGTTTTTATT ACTCTAGGCCGACGA

Insert
5
 Primer Design for PCR-Generated Vector and Insert
For the purposes of primer design, the vector and the insert may be viewed as
two PCR fragments that have to be assembled into a circular DNA molecule.
This means that the primer design rules described above may also be applied
for generation of the vector fragment and the insert fragment sharing overlap-
ping ends. Use the in silico-created final sequence file as a template to design
overlapping primers between the vector and the insert by accomplishing the
same steps as described above, as shown in Figure 2B.
If you intend to use PCR-generated vector for one specific insertion, then the
overlap sequence may be split between the vector and the insert in any combi-
nation to make shorter primers (Figure 2B, step III, overlap shown in orange).
However, if the same PCR-generated vector will be used for assembly of vari-
ous inserts, then the entire overlap sequence must originate from the vector
sequence and must be added to primers that will be used to amplify the insert
(Figure 2B, step III, overlap shown in blue). The latter case is also illustrated
in Figure 3 for assembly of the lacZ gene into a pET21a vector. The pET21a
forward primer (orange arrow) and the reverse primer (blue arrow) start at
the position where the lacZ gene must be inserted. Both vector-specific prim-
ers completely match the vector sequence on the respective strands marked
in orange and blue. This inverse PCR strategy yields a linear vector fragment.
Generally, 10–100 pg of a vector is recommended as a template in the inverse
PCR reaction.
To amplify the lacZ gene, both forward and reverse lacZ-specific priming
sequences (gray) at their 5´ end are fused with 20-nt vector sequences to be
used as overlap sequences in assembly with the vector. Within the lacZ Forward
PCR primer, the overlap sequence (orange) is identical to the 20-nt terminal
sequence on the top strand (orange) of the vector’s left-arm (in the 5´→3´
direction). Within the lacZ Reverse PCR primer, the overlap sequence (blue) is
identical to the 21-nt terminal sequence on the bottom strand (blue) of the vec-
tor’s right-arm (in the 5´→3´ direction). The length of the overlap sequence is
determined by the number of nucleotides needed to reach a Tm ≥ 48°C. If nec-
essary, one may add additional nucleotides between the overlap sequence and
the lacZ-specific sequence, for example, to introduce a unique restriction site.

6
Figure 3: Primer Design for Vector pET21a and lacZ Gene Assembly.

A. pET21a Fragment from PCR

Forward Primer to Amplify pET21a

20-nt Overlap Region 5´-TGAGATCCGGCTGCTAACAAAG-3´

5´-ATTTTGTTTAACTTTAAGAAGGAGATATACAT-3´ TGAGATCCGGCTGCTAACAAAG-3´
3´-TAAAACAAATTGAAATTCTTCCTCTATATGTA-5´ ACTCTAGGCCGACGATTGTTTC-5´

3´-TAAAACAAATTGAAATTCTTCCTCTATATGTA-5´ 21-nt Overlap Region

Reverse Primer to Amplify pET21a

B. lacZ Gene Fragment from PCR

lacZ Forward PCR Primer
20-nt Overlap Region N-terminal Sequence of lacZ Gene

5´-TTTAAGAAGGAGATATACATATGACCATGATTACGGATTC

lacZ Gene
TCAACCAGACCACAGTTTTTACTCTAGGCCGACGATTGTTT- 5´

C-terminal Sequence of lacZ Gene 21-nt Overlap Region

lacZ Reverse PCR Primer

7
 Primer Design for Assembly of Restriction Enzyme Digested Vector and
PCR-Generated Insert.
Restriction enzyme-treated vectors can have 5´-overhangs, 3´-overhangs or
blunt ends. When vector is linearized by restriction digestion, the entire over-
lap sequence must originate from the vector sequence and must be added
to primers that will be used to amplify the insert. The overlap region of the
forward primer for the gene of interest (orange) should line up with the 3´
end of the overhang on the vector’s left arm and extend back until the Tm ≥
48°C (Fig.4A, Left side). This primer also includes gene-specific sequence at
the 3´-end (gray). Keep in mind that the restriction site, which was used to
digest the vector, will be lost in the assembled product. However, additional
nucleotides may be added between the overlap region and gene-specific
sequence region to restore the pre-existing restriction site, or to introduce a
new, unique restriction site. A similar principle is applied to the design of the
reverse primer for the gene of interest (Fig. 4A, Right side).

Figure 4A: Assembly of Restriction Enzyme-Digested Vector and PCR-

derived Insert
Gene-specific Forward PCR Primer

Overlap Region (≥16 nt) Gene-specific Sequence (18-25 nt)

Left Arm Right Arm

5´ Overhang 5´ Overhang
Restriction Enzyme Digested Vector

Restriction Enzyme Digested Vector

Overlap Region (≥16 nt) -3´ 5´-
-5´ 3´- Overlap Region (≥16 nt)

3´ Overhang 3´ Overhang
Overlap Region (≥16 nt) -3´
Gene of Interest 5´-
-5´ 3´- Overlap Region (≥16 nt)

Blunt-end Blunt-end
Overlap Region (≥16 nt) -3´ 5´-
-5´ 3´- Overlap Region (≥16 nt)

Gene-specific Sequence (18-25 nt) Overlap Region (≥16 nt)

Gene-specific Reverse PCR Primer

Figure 4B shows primer design for assembly of the lacZ gene and pMAL-c5X,
digested with NcoI and SbfI. In this example, the forward primer of the gene
has a "C" nucleotide (underlined) inserted between the 18-nt overlap and the
N-terminal sequence of the lacZ gene to ensure the lacZ protein is in frame
with the maltose binding protein.

8
Figure 4B: Primer Design for lacZ Gene and NcoI/SbfI-cut pMAL-c5X
Assembly
lacZ Gene Forward PCR Primer
18-nt Overlap Region N-terminal Sequence of lacZ Gene

5´-AAGGATTTCACATATGTCCATGACCATGATTACGGATTC

5´-GGAAGGATTTCACATATGTC-3´ 5´-GGTAATTAAATAAGCTTC-3´
3´-CCTTCCTAAAGTGTATACAGGTAC-5´
lacZ gene 3´-ACGTCCATTAATTTATTCGAAG-5´

TCAACCAGACCACAGTTTTTACTACGTCCATTAATTTATTCGA- 5´

C-terminal Sequence of lacZ Gene 20-nt Overlap Region

lacZ Gene Reverse PCR Primer

Useful Recommendations for Vector Digestion with Restriction Enzymes

In general, the cloning vector can be linearized by any restriction endonuclease
or by any combination of two restriction endonucleases displaying unique site(s)
at the desired locations within the vector sequence.
Note: Double-digestion of vector DNA with two restriction endonucleases is the
best approach to reduce the uncut vector background.
• Some restriction endonucleases cannot efficiently digest supercoiled DNA
and thus may leave behind different amounts of uncut vector DNA. If not
gel-purified, the uncut vector is transformable and will show up after trans-
formation of the Gibson Assembly reaction, thereby, reducing the overall
fraction of recombinant clones. A table “Cleavage of Supercoiled DNA”
found at www.neb.com/nebecomm/tech_reference/restriction_enzymes/
may be used as a reference for choosing the most suitable restriction endo-
nucleases and the number of activity units required for complete digestion
of plasmid vector.
• Restriction endonucleases might have a reduced activity on plasmid DNA
purified using various plasmid purification kits. In such cases, the extended
restriction time or increased enzyme concentration may be necessary to
digest plasmid vector to completion (or as nearly as possible to comple-
tion). When applicable, NEB highly recommends using High Fidelity (HF)
restriction endonucleases to avoid star activity which may occur when
digesting DNA with elevated amounts of regular restriction endonuclease
for extended periods of time.
• Purification of restriction endonuclease-digested vector is not necessary
unless the same restriction site is present in insert DNA. In such cases,
either heat-inactivate restriction endonuclease before Gibson Assembly
reaction or purify the linearized vector, either by phenol-chloroform extrac-
tion/alcohol precipitation or by electrophoresis on an agarose gel.

9
 Useful Recommendations for PCR Amplification
NEB recommends using Q5 High-Fidelity DNA polymerase (NEB #M0491)
or related products (NEB #M0493 or NEB #M0494) to amplify fragments
of interest prior to assembly. The use of this high-fidelity DNA polymerase
yields PCR products with blunt ends, thereby reducing the error rates at the
fragment junctions.
• When using circular plasmid DNA as a template, it is important to use a
minimal amount of DNA (usually recommended 0.1–0.5 ng of plasmid
template per 50 µl PCR reaction) in order to reduce the template back-
ground after transformation. If higher amounts of plasmid template must
be used in PCR reaction or higher amounts of PCR product must be used
in the Gibson Assembly reaction, it is recommended to digest the PCR
product with DpnI restriction endonuclease in order to destroy plasmid
template before setting up the Gibson Assembly reaction (for protocol
see below).
• Verify PCR product purity and yield by gel electrophoresis. If non-specific
DNA fragments are obtained, you will need to purify the target fragment
from the agarose gel to ensure the correct product assembly during the
Gibson Assembly reaction.
• PCR product purification is not necessary as long as the product is
> 90% pure. You can add unpurified PCR product directly from the PCR
reaction into the Gibson Assembly for up to 20% of the total Gibson
Assembly reaction volume (i.e. PCR products should account for 4 µl, or
less, in a 20 µl Gibson Assembly reaction). Larger volumes of unpurified
PCR products could significantly inhibit both the Gibson Assembly and
the transformation. In such cases, it is recommended to column-purify
PCR products and, if necessary, to concentrate DNA by ethanol precipita-
tion.

(Optional) DpnI Digestion Protocol

When higher amounts of plasmid template must be used in the PCR reac-
tion, it is recommended to digest the PCR product with DpnI (NEB #R0176)
restriction endonuclease in order to destroy plasmid template before setting
up the Gibson Assembly reaction. DpnI cleaves only E. coli Dam methylase-
methylated plasmid DNA, but does not cleave the PCR product since it is not
methylated.
DpnI Digestion Protocol:
1. In a total 10 µl reaction, mix 5–8 μl of PCR product with 1 μl of 10X
NEBuffer 4 and 1 μl (20 units) of DpnI.
2. Incubate at 37°C for 30 minutes.
3. Heat-inactivate DpnI by incubating at 80°C for 20 minutes.
4. Proceed with the Gibson Assembly Cloning procedure, described on
page 11.
10
Gibson Assembly Reaction:
Optimal Quantities
NEB recommends a total of 0.02–0.5 pmols of DNA fragments when 1 or 2
fragments are being assembled into a vector. Efficiency of assembly decreases
as the number or length of fragments increases. To calculate the number of
pmols of each fragment for optimal assembly, based on fragment length and
weight, we recommend the following formula:
pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons)
50 ng of 5000 bp dsDNA is about 0.015 pmols.
50 ng of 500 bp dsDNA is about 0.15 pmols.
The mass of each fragment can be measured using the NanoDrop instrument,
absorbance at 260 nm or estimated from agarose gel electrophoresis followed
by ethidium bromide staining.

Assembly Protocol:
1. Set up the following reaction on ice:

Recommended Amount of Fragments Used for Assembly

2–3 Fragment 4–6 Fragment
Assembly Assembly Positive Control**
Total Amount of 0.02–0.5 pmols* 0.2–1 pmols* 10 µl
Fragments X µl X µl
Gibson
Assembly Master
Mix (2X) 10 µl 10 µl 10 µl
Deionized H2O 10-X µl 10-X µl 0
Total Volume 20 µl*** 20 µl*** 20 µl
* Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts.
Use 5 times more of inserts if size is less than 200 bps.
** Control reagents are provided for two experiments.
*** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may
be required.

2. Incubate samples in a thermocycler at 50°C for 60 minutes. Following

incubation, store samples on ice or at –20°C for subsequent
transformation.

11
 Chemically Competent Cells Transformation Protocol:
1. Thaw chemically competent cells on ice.
2. Transfer 50 μl of competent cells to a 1.5 ml microcentrifuge tube (if
necessary).
3. If the chemically competent cells are from New England Biolabs, add 2 µl
of assembled product to NEB competent cells and go to step 4 directly. If
competent cells are purchased from other manufacture, dilute assembled
products 4-fold with H2O prior transformation. This can be achieved by
mixing 5 µl of assembled products with 15 µl of H2O. Add 2 μl of the diluted
assembled product to competent cells.
4. Mix gently by pipetting up and down or flicking the tube 4–5 times. Do not
vortex. Place the mixture on ice for 30 minutes. Do not mix.
5. Heat shock at 42°C for 30 seconds.* Do not mix.
6. Transfer tubes on ice for 2 minutes.
7. Add 950 μl of room temperature SOC media* to tubes.
8. Place the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
9. Warm selection plates to 37°C.
10. Spread 100 μl of the cells onto the plates with appropriate antibiotics. Use
Amp plates for positive control sample.
11. Incubate plates overnight at 37°C.
* Please note: Follow the manufacturer's protocols for the duration and
temperature of the heat shock step, as well as the optimal medium for
recovery. Typically, transformation of our positive control assembly product
will yield more than 100 colonies on an Amp plate with greater than 80%
colonies containing inserts.

Electrocompetent Cells Transformation Protocol:

1. Thaw electrocompetent cells on ice.
2. Transfer 50 μl of electrocompetent cells to a pre-chilled electroporation
cuvette with 1 mM gap.
3. Dilute assembled products 3-fold with H2O prior electroporation. This can be
achieved by mixing 5 µl of assembled products with 10 µl of H2O. Add 1 μl of
the diluted assembly product to electrocompetent cells.
4. Mix gently by pipetting up.
5. Once DNA is added to the cells, electroporation can be carried out
immediately. It is not necessary to incubate DNA with cells.

12
6. Add 950 μl of room temperature SOC media to the cuvette immediately
after electroporation.
7. Place the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or
rotate.
8. Warm selection plates to 37°C.
9. Spread 100 μl of the cells onto the plates.
10. Incubate overnight at 37°C.

Usage Notes:
To ensure the successful assembly and subsequent transformation of
assembled DNAs, NEB recommends the following:
• Cells: Transformation efficiency of competent cells can vary by several logs.
Perceived assembly efficiency directly correlates to the competence of the
cells used for transformation.
• Electroporation: Electroporation can increase transformation efficiency by
several logs. When using the Gibson Assembly Master Mix product for
electroporation, it is necessary to dilute the reaction 3-fold and use 1 µl for
transformation.
• DNA: Purified DNA for assembly can be dissolved in ddH2O (Milli-Q® water
or equivalent is preferable), TE or other dilution buffers.
• Insert: When directly assembling fragments into a cloning vector, the
concentration of assembly fragments should be 2–3 times higher than
the concentration of vector. For assembly of 3 or more fragments, we
recommend using equilmolar ratio of fragments.
• Biology: Some DNA structures, including inverted and tandem repeats,
are selected against by E. coli. Some recombinant proteins are not well
tolerated by E. coli and can result in poor transformation or small colonies.

13
 Frequently Asked Questions (FAQs):
What should I do if my assembly reaction yields no colonies, a small
number of colonies, or clones with the incorrect insert size following
transformation into E. coli?
Assemble and transform the positive control provided with the Gibson
Assembly Master Mix (see page 11,12). Successful assembly of a positive
control will demonstrate that the assembly mixture is functional and the
transformation conditions are suitable.
Analyze the reaction on an agarose gel. An efficient assembly reaction will
show assembled products of the correct size and the disappearance of
fragments.
Check the primer design of the overlapping DNA fragments to ensure that
there is sufficient overlap to facilitate assembly.
Consider whether the cloned insert may be toxic to E. coli and a low-copy
vector, such as a BAC, should be used. Because the assembled product is a
covalently closed molecule, it may be alternatively amplified by PCR or RCA.

What are the advantages of this method compared to traditional cloning

methods?
Gibson Assembly does not rely on the presence of restriction sites within
a particular sequence to be synthesized or cloned. Therefore, the user has
complete control over what is assembled. Furthermore, the inclusion of
unwanted additional sequence, often used to facilitate the manipulation of
multiple DNA sequences, can be avoided. Lastly, a greater number of DNA
fragments can be joined in a single reaction with greater efficiency than
conventional methods.

How large a DNA fragment can I assemble?

Gibson Assembly has been used to assemble and clone 300 kb DNA fragments
in E. coli, the approximate upper limit for cloning into E. coli. Products as large
as 1 Mb have been assembled using this approach.

How many fragments of DNA can be assembled in one reaction?

The number of DNA segments that can be assembled in one reaction is
dependent on the length and sequence of the fragments. Gibson Assembly has
been used to efficiently assemble up to twelve 0.4 kb inserts into a vector at
one time. However, we recommend the assembly of five or fewer inserts into
a vector in one reaction in order to produce a clone with the correct insert.
A strategy involving sequential assembly can be used if all of the fragments
cannot be assembled in a single reaction.

14
How can I reduce the number of vector-only background colonies?
To significantly reduce the background of unwanted vector-only colonies,
the vector should be a PCR product rather than a restriction fragment. If
background continues to be a problem, the PCR-amplified vector can be
treated with DpnI to remove the template carry-over, if applicable, extracted
from an agarose gel following electrophoresis.

Can you PCR-amplify the assembled product?

Yes. The assembled DNA molecule is covalently joined and may be PCR-
amplified. Additionally, if the final product is a closed circular DNA molecule, it
may be used as a template in rolling-circle amplification (RCA).

What type of competent cells are suitable for transformation of DNA

constructs created using Gibson Assembly?
The resulting DNA constructs are compatible with most E. coli competent
cells. NEB recommends using NEB 5-alpha Competent E. coli (High
Efficiency, NEB #C2987). If the assembled products are larger than 10 kb,
NEB recommends using NEB 10-beta Competent E. coli (High Efficiency, NEB
#C3019) or NEB 10-beta Electrocompetent E. coli (NEB #C3020).

Is this method applicable to the assembly of repetitive sequences?

Yes. However, one must ensure that each DNA fragment includes a unique
overlap so that the sequences may anneal and are properly assembled. The
repetitive sequence can also be internalized in the first stage of a two-stage
assembly strategy. If having repetitive sequences at the ends of each fragment
is unavoidable, the correct DNA assembly may be produced, albeit at lower
efficiency than other, unintended assemblies.

What are the shortest overlaps that can be used with this assembly
method?
Productive assembly has been shown for DNA fragments with as little as
12 bp overlap. However, it depends on the GC content of the overlap. We
recommend using 16 bp overlaps or more for dsDNA assembly with a Tm >
48°C (AT pair = 2°C and GC pair = 4°C).

What are the longest overlaps that can be used with this method?
The quantity of exonuclease in the Gibson Assembly Master Mix has been
optimized for the assembly of DNA molecules with ≤ 100-bp overlaps.

Is it necessary to gel-extract restriction fragments or PCR products?

Gel-extraction of restriction fragments is generally not necessary. A column
cleanup kit or a standard phenol-chloroform extraction, followed by ethanol
precipitation is sufficient. We have also used unpurified PCR products directly
in assembly reactions as long as the PCR product is > 90% pure.
15
 Can ≤ 200 bp dsDNA fragments be assembled by this method?
Yes. For optimal results, use these fragments in ≥ 5-fold excess.

Can ssDNA oligonucleotides be combined and assembled with dsDNA

fragments?
Yes. However, the optimal concentration of each oligonucleotide should
be determined. As a starting point, we recommend using 45 nM of each
oligonucleotide that is less than or equal to twelve 60-base oligonucleotides
containing 30-base overlaps.

Can longer or shorter incubation times be used?

Yes. The Gibson Assembly Kit has been optimized for assembly in 1 hour at
50°C. However, in some cases, 15 minutes may be sufficient. Reaction times
less than 15 minutes are generally not recommended. Extended incubation
times (up to 4 hours) have been shown to improve assembly efficiencies in
some cases.

Will the reaction work at other temperatures?

The reaction has been optimized at 50°C, but it has been shown to work
between 40°C and 50°C.

I would like to produce overlapping dsDNA fragments by PCR. Do I need to

use PCR primers that have been purified by PAGE or HPLC?
No. Standard, desalted primers may be used.

I would like to assemble ssDNA oligonucleotides into dsDNA fragments. Do

I need to use oligonucleotides that have been purified by PAGE or HPLC?
No. Standard, desalted primers may be used.

Can I use a 15-nt overlap that is entirely composed of His-tag repeats (i.e.
CACCACCACCACCAC)?
No, after the His-tag, you must include at least 3 nucleotides, that are not part
of the His-tag repeating sequence. Avoid repeating sequences at the end of an
overlap.

Is it necessary to inactivate restriction enzymes after vector digestion?

No, unless the insert also carries the restriction site that was used to linearize
the vector. You may still linearize the vector with such restriction enzymes,
but it is necessary to heat inactivate the restriction enzyme before mixing
the linearized vector with the insert in Gibson Assembly. If a heat-resistant
restriction enzyme was used to linearize the vector, then vector should be
purified by DNA columns, phenol-chloroform extraction or extracted from
agarose gel after electrophoresis, before coming into contact with the insert.

16
Are there any differences between the Gibson Assembly Master Mix (NEB
#E2611) and Gibson Assembly Master Mix included in the Gibson Assembly
Cloning Kit (NEB #E5510)?
The protocol for the Gibson Assembly Cloning Kit (NEB #E5510) is optimized
to perform one to two fragment assembly and cloning into a vector, while
the protocol for Gibson Assembly Master Mix (NEB #E2611) is optimized
to perform multiple-fragment assemblies (up to six fragments). The major
differences between the two protocols are the length of overlapping sequences
between the adjacent fragments and the incubation time of the assembly
reaction. The 15 minute assembly reaction protocol provided with the Gibson
Assembly Cloning Kit (NEB #E5510) is recommended for assembly of
fragments that are flanked by 15-25 nt overlaps. The Gibson Assembly Master
Mix (NEB #E2611) is recommended for assembly of up to six fragments that
are flanked by 20-80 nt overlaps, and requires a 60 minute reaction time.

Troubleshooting
Positive Control Yields No Colonies Following Transformation into E. coli
• Be sure to perform a positive control assembly reaction with 2X Gibson
Master Mix, followed by transformation using competent cells from (NEB
#C2987) as described on page 11,12. If competent cells are purchased
from other manufacture, dilute assembled products 4 times with H2O
prior transformation. This can be achieved by mixing 5 µl of assembled
products with 15 µl of H2O. Add 2 μl of the diluted assembled product to
competent cells.
• Competent cells may be thawed only once and cannot be repeatedly
frozen and thawed without extreme loss in competency. Cells are best
thawed on ice and DNA added as soon as the last bit of ice in the tube
disappears.
• Do not vortex competent cells. Mix cells and DNA by gently pipetting
up and down. Check cell competency by transforming 100 pg of pUC19
plasmid provided with the kit. Expect 1–3 x 109 colonies formed/μg DNA
after overnight incubation on LB-ampicilin plates at 37°C.

Gibson Assembly Reaction Yields No Colonies Following

Transformation into E. coli.
• Assemble and transform the positive control provided with the Gibson
Assembly Cloning Kit. Successful assembly of a positive control will
demonstrate that the Gibson Assembly Master Mix is functional and the
transformation conditions are suitable.
• Check the primer design of the overlapping DNA fragments to ensure that
there is sufficient and correct overlap to facilitate assembly.

17
 • Avoid overlaps with highly palindromic sequences as they may cause
up to a 10-fold reduction in recombinant colonies. When assembling
fragments into a multiple cloning site (MCS) of a cloning vector, it is
strongly recommended to use restriction sites that are located on the
edges of the MCS to avoid overlap regions with highly palindromic
sequences. Plate higher amounts of transformation reaction when using
restriction sites that are located in the middle of the MCS of the cloning
vector.
• Repeat the Gibson Assembly reaction using higher concentrations
of fragments and/or vector. Make sure that the total volume of PCR-
amplified products does not exceed 20% of Gibson Assembly reaction. If
necessary, purify PCR fragments and/or PCR-amplified vector before the
assembly reaction.
• Some DNA structures, including inverted and tandem repeats, are
selected against by E. coli. Some recombinant proteins are not well toler-
ated by E. coli and can result in poor transformation.
• Test the success of the Assembly by performing PCR with primers that
flank the assembled product.
• Consider whether the cloned insert may be toxic to E. coli, and a low-
copy vector, such as a BAC, should be used. Because the assembled
product is a covalently-closed molecule, it may be alternatively amplified
by PCR or rolling-circle amplification (RCA).

Gibson Assembly Reaction Yields High Number of Clones

with Incorrect Inserts
• Make sure that your PCR product is a single band of the correct size. If
the PCR product is contaminated with non-specific bands, it is necessary
to gel-purify the PCR product to ensure cloning of the correct insert.
• Consider whether the cloned insert may be toxic to E. coli and a low-copy
vector, such as a BAC, should be used. Because the assembled product is
a covalently closed molecule, it may be alternatively amplified by PCR or
RCA.

Gibson Assembly Reaction Yields High Number of Small Colonies

• Some recombinant proteins are not well-tolerated by E. coli and can
result in poor transformation efficiency or small colonies. Use a low copy
number vector (i.e. pACYC184) or a vector with tight control of protein
expression. When assembling into the pUC19 vector, make sure that your
gene is not in frame with lacZ alpha fragment.

18
Gibson Assembly Reaction Yields a High Number of Clones
without the Insert
• PCR products may carry over large quantities of uncut plasmid template.
To remove plasmid template, treat PCR products with DpnI restriction
endonuclease before performing Gibson Assembly. Protocol for DpnI
digestion can be found on page 10.
• Restriction enzyme digested vector may carry over large quantities
of uncut plasmid. Some restriction enzymes do not cut supercoiled
plasmids to completion. The best way to reduce uncut vector background
is to digest the vector with two different restriction endonucleases. If a
single enzyme must be used, avoid restriction enzymes that leave four-
base single-stranded overhangs rich in C/G (i.e. CCGG overhang). These
overhangs may self-anneal to form the transformable form of the vector
molecule. Also, increase units and/or incubation time and/or purify the
linear vector from agarose gel.

References:
1. Gibson, D.G. et.al. (2009) Nature Methods, 343–345.
2. Gibson, D.G. et al. (2010) Nature Methods, 901–903.

For additional references and information on Gibson Assembly Master Mix, visit
www.syntheticgenomics.com. and www.jcvi.org.

19
 Ordering Information
PRODUCT NEB # SIZE
Gibson Assembly Master Mix E2611S/L 10/50 reactions
COMPANION PRODUCTS

Gibson Assembly Cloning Kit E5510S 10 reactions

Q5 High-Fidelity DNA Polymerase M0491S/L 100/500 units

Q5 High-Fidelity 2X Master Mix M0492S/L 100/500 rxns

Q5 Hot Start High-Fidelity DNA Polymerase M0493S/L 100/500 units

Q5 Hot Start High-Fidelity 2X PCR Master Mix M0494S/L 100/500 rxns

6 x 0.2 ml/
NEB 5-alpha Competent E. coli (High Efficiency) C2987I/H
20 x 0.05 ml
6 x 0.2 ml/
NEB 10-beta Competent E. coli (High Efficiency) C3019I/H
20 x 0.05 ml

NEB 10-beta Electrocompetent E. coli C3020K 6 x 0.1 ml

SOC Outgrowth Medium B9020S 4 x 25 ml

20
21
 DNA CLONING
DNA AMPLIFICATION & PCR
EPIGENETICS
RNA ANALYSIS
LIBRARY PREP FOR NEXT GEN SEQUENCING
PROTEIN EXPRESSION & ANALYSIS
CELLULAR ANALYSIS

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