A Takara Bio Company

Steve Oh1, Gia Jokhadze, Tommy Duong, Magnolia Bostick, Andrew A. Farmer

Abstract

1 Corresponding Author
Clontech Laboratories, Inc., 1290 Terra Bella Ave., Mountain View, CA 94043

pU

Ac
30

TRP1

00

Accuracy

286

Junction 1

Three-Insert Multiple-Fragment Cloning

Vector + insert
Negative control (no insert)
Cloning accuracy

Results

635 colonies

401 colonies

1 colony

39 colonies

Junction 4

Junction 6

96% (25/26 correct colonies)

pU

n
igi
r
o

MBP

TRP

GGATGTTTTGGCTCTGGTCAATGATTACGGCATTGATAT
ACGGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGA

89 colonies

111 colonies

392 colonies

1 colony

39 colonies

78 colonies

100% (26/26 correct colonies)

19% (5/26 colonies)

73% (19/26 correct colonies)

er
t4

Results

3500

in

TR
250 0

High cloning accuracy is obtained when cloning single and multiple inserts with In-Fusion Cloning. In-Fusion HD Cloning and Gibsons
method were each used to clone single and multiple (3) inserts into puC19 (linearized with BamHI). Each cloning system was tested
under the recommended conditions of its own cloning protocol. In an effort to make a more direct comparison with In-Fusion
cloning, this multiple-insert experiment with Gibsons enzyme mix was also run at the shorter In-Fusion reaction time. All cloning
reactions were then transformed into Stellar Competent Cells, and 1/10th of each reaction was plated. Colony counts and clone
sequence verification were used to evaluate the results of each reaction. The results of single-insert cloning were roughly
comparable between the two systems, but the In-Fusion Cloning technology distinguished itself with lower levels of background
an asset seen to even greater extent in the multiple-insert experiment, where it was also found to have much higher cloning accuracy
than Gibsons method.

5
insert

00

Cloning accuracy

TRP

5-insert_lacZ-MBP-TRP1
4583 bp

30

Negative control (no insert)

1,592

AAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTACGA

MBP

Vector + insert

Gibsons Method
Incubate at 50C for 15 min
Incubate at 50C for 60 min
Results

++++

50
mo r
0
lac pro latceo
pelraacZ
to
inse r
rt 1

3. Gibson, D. G.,et al. (2009) Nature Methods 6(5):343345

Results

263

LacZ

MBP

MBP

MBP

TRP

Vector

4500

Am

2. Lestini, R., et al. (2013) Nucleic Acids Research 41(22):1035810370

Conditions

In-Fusion HD Cloning
Incubate at 50C for 15 min

+++

AGAACCGTACTTCACCTGGCCGCTGATTGC
TCAACCTGCAAGAACCGTACTTCAC

2
ert
ins

1. Chen, C. G., et al. (2014) Nucleic Acids Research 42(4):e26

3-insert,
multi-fragment

Multiple Insert Cloning

For Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale. Takara and the Takara logo
are trademarks of TAKARA HOLDINGS, Kyoto, Japan. Clontech, the Clontech logo, In-Fusion, and Stellar are trademarks
of Clontech Laboratories, Inc. All other marks are the property of their respective owners. Certain trademarks may not
be registered in all jurisdictions. Clontech is a Takara Bio Company. 2014 Clontech Laboratories, Inc.

MBP

40
0

100% (26/26 correct colonies)

Number of Colonies per Plate

0
100

References

Results

209

MBP

Single Insert Cloning

Conditions

When cloning multiple fragments in a single In-Fusion reaction, increasing the overlap
between fragments from 15 bp to 20 bp improves cloning efficiency ~5- to 6-fold.

ATCTGGATTAACGGCG
TCTGGATTAACGGCGATAAAGGCTA
ATAAAGGCTA
AAGGTAAACTGGTAA
AAGGTAAACTGGTAATCTGGATTAACGGCG

Junction 5

Gibsons Method
Incubate at 50C for 15 min

242

GTGAGTAAGCTCACTCATTAGGCACCCCAGGCTTTACACT

Junction 3

In-Fusion HD Cloning
Incubate at 50C for 15 min

Vector only

Vector

Junction 2

In-Fusion Cloning is more accurate than Gibsons method when multiple inserts
are cloned simultaneously.

1/20

Five-Insert Multiple-Fragment Cloning

Increasing the length of overlaps from 15 bp to 20 bp improves the efficiency of multiple-fragment cloning.

Conclusions

8.5/10

91.5%

Fast, easy cloning without ligation. Panel A. In-Fusion Cloning is a complete, optimized system that allows ligationindependent, directional cloning of PCR products into any vector, at any site of linearization in just one step. Your linearized vector
is combined with your PCR-amplified insert and the In-Fusion enzyme mix. The cloning reaction takes as little as 15 minutes and is
immediately ready for transformation into competent cells without further treatment. This powerful cloning technology is seamless,
accurate, and adaptable to a wide range of applications, including multiple-fragment cloning, large-fragment cloning, site-directed
mutagenesis, the addition of small tags to your gene of interest, direct cloning into large expression vectors, and high-throughput
experiments. Panel B. The In-Fusion Cloning system has been successfully used by many high-throughput users worldwide.
This bar graph shows the percentage of correct clones obtained during generation of an ORF collection by the Harvard Institute
of Proteomics. 3,367 individual clones were generated in order to obtain the desired 2,000 ORFs, with cloned inserts ranging
in size from 5003,500 bp. Full-length sequencing of the inserts determined that over 3,000 of these clones contained the correct
insert, delivering over 91% cloning accuracy.

In-Fusion reaction

1/20

15-bp overlap
(blue/white)

20-bp overlap
(blue/white)

Dilution

1/10

1/10

Vector only

0/5

0/5

5-insert,
multi-fragment

21/5

115/12

Incorrect insert

Mutations

3,081

15 0

Correct insert

8.5/10

Increasing the length of fragment overlaps from 15 bp to 20 bp improves cloning efficiency for three-insert multiple-fragment cloning.
Panel A. 1.1 kb, 700 bp, and 600 bp inserts (20 ng each) were cloned into a 2.5 kb linearized vector (100 ng). 1/20th, 1/10th and the remainder
of the cloning reactions were plated. Panel B. Parallel experiments were performed with either 15-bp or 20-bp fragment overlaps.
15 bp was determined to be the optimal length for overlaps using the original In-Fusion Cloning kit. We tested whether the optimal
overlap length differed for the current In-Fusion HD enzyme and if this could improve the efficiency of multiple-fragment cloning.
Using 20-bp overlaps improved efficiency by ~6-fold. Plasmids were purified and sequenced. 100% of readable sequences were correct.

Number of Clones

Deletions

20-bp overlap

+++/++++ represent the relative estimation of colonies on plates with too many colonies to count.

00

t3

Multiple
fragments

20

insert 3

15-bp overlap

250 0

Overall Study Results

Small
tags

se

Length of cloned DNA fragments

HTP cloning in collaboration with researchers at Harvard

Single
fragments

15 0 0

rt 2

20

Number of Colonies per Plate

Dilution

3-insert_MBP-AcGFP-TRP1
4871 bp

ins e r

Construct

or

400
0

40

12
0

While In-Fusion Cloning simply and accurately clones single inserts into any vector
of choice, cloning applications are becoming increasingly more complex. In traditional
cloning systems, increasing the number of fragments can have a detrimental effect
on both efficiency and accuracy. However, In-Fusion Cloning maintains the high
accuracy typical of single-insert cloning even under more challenging demands,
by taking advantage of the host organisms highly evolved repair machinery. Here,
we improved cloning efficiency of multiple-insert cloning by extending the overlapping
sequence (see image below), and increasing the stability between fragments.

60

Amp

15 bp overlap
between insert
and vector

80

350 0

Introduction

Any
vector

lac prom
ote
r

00
45
n
igi

in

PCR

100

0
100

Any
insert

0
501
75
0
751
1,
00
0
1,0
01
1,2
50
1,2
51
1,5
00
1,5
01
1,7
50
1,7
51
2,0
00
2,0
01
2,2
50
2,2
51
2,5
00
2,5
01
2,7
50
2,7
51
3,0
00
3,0
01
3,2
50
3,2
51
3,5
00

Complete System

GF

Overlaps were Increased from 15 bp to 20 bp

for Three-Insert Multiple-Fragment Cloning

201
50

In-Fusion Cloning: Seamless,

Ligation-Independent Cloning

rt 1
se
in
BP
M

In-Fusion Cloning technology enables directional, seamless cloning of any PCRamplified insert into any linearized vector in a single 15-minute reaction. No additional
treatment of the PCR fragment is required (such as restriction digestion, ligation,
phosphorylation, or blunt-end polishing). Instead, the In-Fusion enzyme generates
short regions of single-stranded overlaps between vector and insert(s), facilitating
accurate, directional cloning of the desired fragments. This overlap is designed
into the PCR primers used to amplify the desired insert sequences. While cloning
single inserts is an extremely efficient process, cloning experiments are becoming
increasingly challenging. Multiple inserts must be cloned, and kept in frame, in order
to build complicated constructs, to stitch genes together from synthetic building
blocks, or to engineer new functionalities through combinatorial protein domain
swapping, etc. Here, we present data showing how multiple-fragment cloning
efficiency and accuracy can be significantly increased by optimizing various
parameters of the In-Fusion Cloning reaction. This further extends the versatility
of the In-Fusion Cloning system to meet the coming challenges of innovative research
in the synthetic biology field.

Percentage of clones with the correct

full-length insert sequence

24

Optimized In-Fusion Cloning System Demonstrates High Efficiency

and Accuracy of Multi-Fragment Cloning

Clontech Laboratories, Inc.

P1

20

00

Increasing the length of overlaps from 15 bp to 20 bp improves cloning efficiency for five-insert multiple-fragment cloning.
Panel A. Five dsDNA inserts were designed to contain 20-bp overlaps between fragments. A comparison to 15 bp overlaps was tested
by shortening the ends of insert 2 by 5 bp (highlighted in yellow). Panel B. 450480 bp inserts (20 ng each) were cloned into a 2.5 kb
linearized vector (33 ng). 1/10th of the cloning reactions were plated. lacZ (insert 1), which turns cells blue in our system, was included
for rapid identification of positive clones. Panel C. Shortening the ends of just one insert decreased the cloning efficiency by ~5-fold.
Blue colonies resulting from the 20-bp overlap reactions were further analyzed by colony PCR, and 10/10 were found to contain
a band corresponding to the five-insert product (data not shown).

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