Takara Bio USA, Inc.

In-Fusion® Snap
Assembly User Manual

Cat. Nos. 638943–638949, 638951, 638952

(060822)

Takara Bio USA, Inc.

2560 Orchard Parkway, San Jose, CA 95131, USA
U.S. Technical Support: [email protected]

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 In-Fusion® Snap Assembly User Manual
Table of Contents
I. Introduction...............................................................................................................................................................3
II. List of Components ...................................................................................................................................................4
III. Additional Materials Required ...................................................................................................................................5
IV. PCR Fragment Amplification and Experimental Preparation......................................................................................5
A. Preparation of a Linearized Vector.........................................................................................................................5
B. PCR Primer Design ...............................................................................................................................................5
C. PCR Amplification of Target Fragment .................................................................................................................6
D. Control Reactions ..................................................................................................................................................6
V. Protocol: In-Fusion Snap Assembly Cloning with Spin-Column Purification .............................................................7
A. Guidelines for Spin-Column Purification of PCR-Amplified Fragments.................................................................7
B. In-Fusion Snap Assembly Cloning with Spin-Column-Purified PCR-Amplified Fragments....................................7
VI. Transformation Procedure .........................................................................................................................................9
Protocol Transformation Using Stellar Competent Cells ................................................................................................9
VII. Expected Results .......................................................................................................................................................9
VIII. Troubleshooting Guide ............................................................................................................................................10
Appendix A. PCR Primer Design ....................................................................................................................................12
Appendix B. pUC19 Linearized Vector Information........................................................................................................15

Table of Figures
Figure 1. In-Fusion Snap Assembly protocol overview. .....................................................................................................4
Figure 2. Universal primer design for In-Fusion technology. ...........................................................................................13
Figure 3. Examples of primers designed for In-Fusion cloning. .......................................................................................14
Figure 4. pUC19 Linearized Vector map and multiple cloning sites (MCS). ....................................................................15

Table of Tables
Table 1. In-Fusion Snap Assembly protocol outline...........................................................................................................3
Table 2. In-Fusion Snap Assembly cloning kit components. ..............................................................................................4
Table 3. Recommended In-Fusion reactions for purified fragments ...................................................................................7
Table 4. Troubleshooting guide for In-Fusion cloning experiments..................................................................................10

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 In-Fusion® Snap Assembly User Manual
I. Introduction
In-Fusion Snap Assembly cloning kits are designed for fast, directional cloning of one or more fragments of
DNA into any vector. The cornerstone of In-Fusion cloning technology is our proprietary In-Fusion Enzyme,
which fuses DNA fragments (e.g., PCR-generated inserts and linearized vectors) efficiently and precisely by
recognizing 15-bp overlaps at their ends. These 15-bp overlaps can be engineered by designing primers for
amplification of the desired sequences. In-Fusion Snap Assembly kits offer increased cloning efficiency over
previous generations of In-Fusion kits, especially for long fragments, short oligonucleotides, and multiple
fragments.

• Clone any insert, into any location, within any vector you choose
• Efficiently clone a broad range of fragment sizes
• Clone multiple DNA fragments simultaneously into any vector in a single reaction
• No restriction digestion, phosphatase treatment, or ligation required
• Final constructs are seamless with no extra or unwanted base pairs

The table below is a general outline of the protocol used for the In-Fusion Snap Assembly cloning kits. This
outline is further illustrated in Figure 1. Please refer to the specified pages for details on performing each step.

Table 1. In-Fusion Snap Assembly protocol outline

Step Action Pages

1 Select a base vector and identify the insertion site. Linearize the
vector by restriction enzyme digestion or inverse PCR and purify. 5

2 Design PCR primers for your gene of interest with 15-bp extensions
(5’) that are complementary to the ends of the linearized vector. 5

3 Amplify your gene of interest with PrimeSTAR® Max DNA

Polymerase. Verify on an agarose gel that your target DNA has been 6
amplified and determine the integrity of the PCR product.

4 Spin-column purify your PCR product. 7

5 Set up your In-Fusion Snap Assembly cloning reaction:

2 μl 5X In-Fusion Snap Assembly Master Mix
X μl Linearized vector 7–8
X μl Insert
X μl dH20 to a total reaction volume of 10 μl. Mix well.
6 Incubate the reaction for 15 min at 50°C, then place on ice. 8

7 Transform competent cells with 2.5 μl of the reaction mixture from

Step 6. 9

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Figure 1. In-Fusion Cloning protocol overview for both liquid and EcoDry™ formats. In-Fusion Snap Assembly utilizes the same
cloning protocol as previous liquid In-Fusion HD cloning kits.

II. List of Components

All In-Fusion Snap Assembly cloning kits contain 5X In-Fusion Snap Assembly Master Mix, linearized pUC19
Control Vector (50 ng/μl), and 2 kb Control Insert (40 ng/μl).
Store all components at –20°C.
Please see the table below for more information on the components included in your kit.
Table 2. In-Fusion Snap Assembly cloning kit components.
NucleoSpin Gel Stellar™ PrimeSTAR Max
Cat.
Product name Size and PCR Clean-Up Competent Cells DNA Polymerase
No.
included included included
In-Fusion Snap
Assembly 638945 10 Rxns Yes Yes Yes
Starter Bundle
In-Fusion Snap
Assembly Value 638946 50 Rxns Yes Yes Yes
Bundle
638947 10 Rxns No No No

638948 50 Rxns No No No

In-Fusion Snap 638949 250 Rxns No No No

Assembly
Master Mix 638943 500 Rxns No No No

638944 1,000 Rxns No No No

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III. Additional Materials Required
The following materials are required but not supplied:
• Ampicillin (100 mg/ml stock) or other antibiotic required for plating the In-Fusion reaction
• LB (Luria-Bertani) medium (pH 7.0)
• LB/antibiotic plates
• SOC medium

IV. PCR Fragment Amplification and Experimental Preparation

A. Preparation of a Linearized Vector
To achieve a successful In-Fusion reaction, you must first generate a linearized vector. The linearized vector can
be generated using restriction enzymes (single or double digests) or by inverse PCR.
Due to differences in cutting efficiencies, different restriction enzymes will generate different amounts of
background. Generally speaking, two different cut sites are better than one for cloning. Efficiency of digestion
will always be better if the restriction sites do not overlap and have at least 5 bases between them. (This varies
with each enzyme, but the majority digest at >90% efficiency in these conditions.) In addition, increasing the
enzyme digestion time and the digestion reaction volume will reduce the background.
Recommendations for preparation of a linearized vector by restriction enzyme digestion:
1. Incubate your restriction digest as directed by the restriction enzyme supplier. For many enzymes, incubation
for several hours can increase linearization and reduce background.
2. After digestion, purify the linearized vector using any available PCR purification kit. We recommend gel
purification using the NucleoSpin Gel and PCR Clean-Up, sold separately (Cat. # 740609.50) and also
included in the In-Fusion Snap Assembly Starter Bundle and Value Bundle (Cat. # 638945 and 638946,
respectively).
3. [Control] Check the background of your vector by transforming competent cells with 5–10 ng of the
linearized and purified vector, in the absence of In-Fusion cloning master mix. If the background is high,
continue digesting the vector for a longer time after the addition of more restriction enzyme(s). Incubate 2 hr
to overnight. Gel-purify the remainder of the vector and transform again.

B. PCR Primer Design

Our online Primer Design tool can easily design primers for amplification of insert fragments, compatible
with either linearization method, as well as vector primers for linearization via inverse PCR:
takarabio.com/in-fusion-tools

If you would like more information about primer design, please refer to Appendix A.

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C. PCR Amplification of Target Fragment
The In-Fusion Cloning method is not affected by the presence or absence of A-overhangs, so you can use
any thermostable DNA polymerase for amplification, including proofreading enzymes. For the best
results, we recommend using our PrimeSTAR Max DNA Polymerase (sold in In-Fusion Snap Assembly
Starter and Value Bundles, and separately as Cat. No. R045A), which provides exceptionally accurate and
efficient DNA amplification. The amount of time required for the extension step has been standardized,
allowing amplification of large amounts of template DNA that would otherwise be difficult to amplify.

1. Amount of template to use with PrimeSTAR Max DNA Polymerase (for a 25-μl reaction, with a 5
sec/kb extension time for genomic DNA, λ DNA, and plasmid DNA templates, or a 5–10 sec/kb
extension time for cDNA templates)
• Human genomic DNA 5 ng–100 ng
• E. coli genomic DNA 100 pg–100 ng
• λ DNA 10 pg–100 ng
• Plasmid DNA 10 pg–1 ng
• cDNA ≤ the equivalent of 25–125 ng total RNA*

*If you are amplifying from a pool of cDNA, the amount of template DNA required depends on the
relative abundance of the target message in your mRNA population

2. PCR product sizes that can be obtained with PrimeSTAR Max DNA Polymerase (with a 5
sec/kb extension time for genomic DNA and λ DNA templates, or a 5–10 sec/kb extension time for
cDNA templates)
• Human genomic DNA up to 6 kb
• E. coli genomic DNA up to 10 kb
• cDNA up to 6 kb
• λ DNA up to 15 kb
3. Analysis of PCR products: When PCR cycling is complete, use an agarose gel to confirm that you
have obtained a single DNA fragment and to estimate the concentration of your PCR product.
Quantify the amount of DNA by measuring against a known standard or DNA mass ladder run on the
same gel.

D. Control Reactions
When using an In-Fusion kit for the first time, we strongly recommend that you perform the positive and
negative control reactions in parallel with your In-Fusion Cloning reaction. The positive control should
consist of a circular vector of known concentration (competent cells should give >2 x 108 cfu/μg), and the
negative control should consist of a known amount of your linearized vector (see Section VII for
Expected Results). Performing the control reactions will verify that the system is working properly. The
2-kb Control Insert included in the In-Fusion Snap Assembly cloning kits has already been purified, so
there is no need for further treatment prior to the cloning reaction.

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V. Protocol: In-Fusion Snap Assembly Cloning with Spin-Column Purification
A. Guidelines for Spin-Column Purification of PCR-Amplified Fragments
Following PCR, verify by agarose gel electrophoresis that your target fragment has been amplified. If a
single band of the desired size is obtained, you can EITHER spin-column purify (see protocol below)
OR treat your PCR product with Cloning Enhancer (Cat. # 639615, Protocol not described in this
document. Please refer to the Cloning Enhancer user manual). However, if non-specific background
or multiple bands are visible on your gel, isolate your target fragment by gel extraction, then spin-column
purify. If you use PCR to amplify your vector and insert and you obtain both a PCR-amplified vector
AND PCR-amplified fragment(s) without nonspecific background, you can use the Cloning Enhancer
protocol.

1. If nonspecific background bands are observed on an agarose gel, isolate your target fragment by gel
extraction, then spin-column purify.
2. Spin-column purify your PCR product (e.g., insert) by using a silica-based purification system,
following the manufacturer’s protocol. We recommend the NucleoSpin Gel and PCR Clean-Up, sold
separately (Cat. # 740609.50) and also included in the In-Fusion Snap Assembly Starter Bundle and
Value Bundle (Cat. # 638945 and 638946, respectively). During purification, avoid nuclease
contamination and exposure of the DNA to UV light for long periods of time.
3. After purification, proceed with the In-Fusion Cloning Procedure for Spin Column-Purified PCR
Fragments (Section V.B).

B. In-Fusion Snap Assembly Cloning with Spin-Column-Purified PCR-

Amplified Fragments
In general, good cloning efficiency is achieved when using 200-ng combined amount of vector and inserts
in a 10-μl reaction, regardless of their lengths. More is not better. If the size of the PCR fragment is
shorter than 0.5 kb, maximum cloning efficiency may be achieved by using less than 50 ng of fragment.
Table 3. Recommended In-Fusion reactions for purified fragments

Negative control Positive control

Reaction component Cloning reaction
reaction reaction
2 μl of 2 kb
Purified PCR fragment 10–200 ng –
control insert
1 μl of pUC19
Linearized vector 50–200 ng 1 μl
control vector
5X In-Fusion Snap Assembly
2 μl 2 μl 2 μl
Master Mix

Deionized Water to 10 μl to 10 μl to 10 μl

In general, for optimal results under standard conditions use an insert to vector molar ratio of 2:1. When
performing a cloning reaction with two or more inserts, the molar ratio of each of the multiple inserts
should still be 2:1 with regards to the linearized vector, i.e., two moles of each insert for each mole of
linearized vector. The molar ratio of two inserts with one vector should be 2:2:1. For more details, please
read the In-Fusion Snap Assembly Multiple-Insert Cloning Protocol-At-A-Glance.
For more detailed guidelines on molar ratios please see the “Tips” subsection under “FAQs” on our In-
Fusion Cloning tips and FAQs page: https://www.takarabio.com/learning-centers/cloning/in-fusion-
cloning-faqs
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 In-Fusion® Snap Assembly User Manual
NOTE: A molar ratio calculator is included in our online cloning tools. The tool currently supports
cloning reactions with up to five inserts: takarabio.com/molar-ratio

1. Set up the In-Fusion Snap Assembly cloning reaction:

5X In-Fusion Snap Assembly
2 μl Master Mix
x μl* Linearized vector
x μl* Purified PCR fragment
x μl dH2O (as needed)
10 μl Total volume
* For reactions with larger combined volumes of vector and PCR insert (>7 μl of vector + insert), double
the amount of enzyme premix, and add dH20 for a total volume of 20 μl.
2. Adjust the total reaction volume to 10 μl using deionized H2O and mix the reaction.
3. Incubate the reaction for 15 min at 50 °C, then place on ice.
NOTE: The In-Fusion reaction is completed within the required 15-min incubation. Longer
incubation times do NOT increase cloning efficiency, even with multiple-insert cloning reactions.
4. Continue to the Transformation Procedure (Section VI). You can store the cloning reactions at
–20°C until you are ready.

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VI. Transformation Procedure
Protocol Transformation Using Stellar Competent Cells
The following protocol has been optimized for transformation using Stellar Competent Cells, sold as a part of
In-Fusion Snap Assembly Starter and Value Bundles and separately in several formats. If you are not using Stellar
Competent Cells, follow the transformation protocol provided with your cells, but you may need to dilute the In-
Fusion reaction mixture prior to transformation to increase cloning efficiency (See Table 4, Troubleshooting Guide).
We strongly recommend the use of competent cells with a transformation efficiency ≥1 x 108 cfu/ug.
For complete information on the handling of Stellar Competent Cells, please see the full Protocol.
1. Thaw Stellar Competent Cells on ice just before use. After thawing, mix gently to ensure even distribution,
and then move 50 µl of competent cells into a 14-ml round-bottom tube (Falcon tube). Do not vortex.
2. Add 2.5 µl of the In-Fusion reaction mixture to the competent cells.
IMPORTANT: DO NOT add more than 5 μl of the reaction to 50 μl of competent cells. MORE IS NOT
BETTER. Using too much of the reaction mixture inhibits the transformation.
3. Place the tubes on ice for 30 min.
4. Heat shock the cells for exactly 45 sec at 42°C.
5. Place tubes on ice for 1–2 min.
6. Add SOC medium to bring the final volume to 500 µl. SOC medium should be warmed to 37°C before using.
7. Incubate by shaking (160–225 rpm) for 1 hr at 37°C.
8. Place 1/100–1/5 of each transformation reaction into separate tubes and bring the volume to 100 μl with SOC
medium. Spread each diluted transformation reaction on a separate LB plate containing an antibiotic
appropriate for the cloning vector (i.e., the control vector included with the kit requires 100 μg/ml of
ampicillin).
NOTE: For cloning reactions with more than two fragments, we recommend plating a larger volume (1/5–1/3
of each transformation reaction).

9. Centrifuge the remainder of each transformation reaction at 6,000 rpm for 5 min. Discard the supernatant and
resuspend each pellet in 100 μl fresh SOC medium. Spread each sample on a separate LB plate containing the
appropriate antibiotic. Incubate all of the plates overnight at 37°C.
10. The next day, pick individual isolated colonies from each experimental plate. Isolate plasmid DNA using a
standard method of your choice (e.g., miniprep). To determine the presence of an insert, analyze the DNA by
restriction digestion or PCR screening.

VII. Expected Results

The positive control plates typically develop several hundred colonies when using cells with a minimum
transformation efficiency of 1 x 108 cfu/μg. The negative control plates should have few colonies.
The number of colonies on your experimental plates will depend on the amount and purity of the PCR product
and linearized vector used for the In-Fusion cloning reaction.
• The presence of a low number of colonies on both plates—typically, a few dozen colonies—
is indicative of either transformation with too much of the reaction, or poor DNA/primer quality.
• The presence of many (hundreds) of colonies on the negative control is indicative of incomplete vector
linearization.

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VIII. Troubleshooting Guide
If you do not obtain the expected results, use the following guide to troubleshoot your experiment. To confirm
that your kit is working properly, perform the control reactions.

NOTE: Many troubleshooting topics are covered in our online In-Fusion Cloning tips and FAQs:
https://www.takarabio.com/learning-centers/cloning/in-fusion-cloning-faqs

Table 4. Troubleshooting guide for In-Fusion cloning experiments

A. No or Few Colonies Obtained from Transformation

Description of 0
Possible explanation Solution
problem

Transformed with too

Do not add more than 5 μl of the In-Fusion reaction to 50 μl of
much In-Fusion
competent cells (see Section VI for details).
reaction
If your cloning efficiency is low, you may obtain better results
Competent cells are if you dilute the reaction. For some cell strains, it may be better
sensitive to the to dilute the In-Fusion reaction with TE buffer 5–10 times prior
Low In-Fusion enzyme to transformation (add 40–90 μl of TE buffer to 10 μl of
transformation In-Fusion reaction).
efficiency
Bacteria were not Check transformation efficiency. You should obtain ≥1 x 108
competent cfu/μg; otherwise use fresh competent cells.

Regions of homology
were not long enough
Increase homologous region of primers from 15 bp to 20 bp.
for efficient cloning of
>2 fragments at once

It is imperative to obtain the highest DNA concentration

possible in your In-Fusion reaction. Either the amount of vector
Low DNA concentration or the amount of PCR fragment was too low. We recommend
in reaction
always using 200 ng combined amount of vector and inserts in
a 10-μl reaction (see Table 3).
If your fragment was gel purified, it is imperative to obtain the
highest DNA concentration possible in your In-Fusion reaction.
Gel purification The total volume of purified vector and insert should not exceed
introduced 5 μl.
Low quality DNA contaminants If possible, optimize your PCR amplification reactions such that
fragments you generate pure PCR products and use Cloning Enhancer
instead (see Section V.A for details).
Repeat PCR amplification and purify product using a different
Suboptimal PCR method of purification. Alternatively, perform phenol:chloroform
product extraction on your original PCR product, followed by ethanol
precipitation.

Check primer sequences to ensure that they provide 15 bases

Primer sequences are
of homology with the region flanking the insertion site (see
incorrect
Section IV).

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B. Large Numbers of Colonies Contained No Insert

Description of
Possible explanation Solution
problem

Incomplete linearization It is important to remove any uncut vector prior to use in the In-
of your vector Fusion reaction. If necessary, recut your vector and gel purify.

If your insert was amplified from a plasmid, closed circular DNA

may have carried through purification and contaminated the
cloning reaction:
Contamination of
To ensure the removal of any plasmid contamination, we
Large numbers of In-Fusion reaction by
recommend linearizing the template DNA before performing
colonies obtained plasmid with same
PCR.
with no insert antibiotic resistance
If you spin-column purify your insert, treating the PCR product
with DpnI before purification will help to remove contaminating
template DNA.

Plates too old or

Be sure that your antibiotic plates are fresh (<1 month old).
contained incorrect
Check the antibiotic resistance of your fragment.
antibiotic

C. Clones Contained Incorrect Insert

Large number of Your PCR product If your PCR product is not a single distinct band, then it may be
colonies contain contained non-specific necessary to gel purify the PCR product to ensure cloning of
incorrect insert sequences the correct insert. See Section V.A for more information.

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Appendix A. PCR Primer Design
In-Fusion allows you to join two or more fragments (e.g., vector and insert or multiple fragments) as long as they
share 15 bases of homology at each end. This homology is achieved through primers designed specifically for
your experiment. Figure 2 outlines the guidelines for primer design discussed below and Figure 3 gives specific
examples of In-Fusion PCR primers.

1. The 5’ end of each primer must contain 15 bases that are homologous to 15 bases at one end of the DNA
fragment to which it will be joined (i.e., the vector or another insert).

NOTE: When joining more than two fragments (including the linearized vector), we strongly recommend
increasing the homologous region to 20 bp. We have found that this modification yields 5- to 7-fold more
transformant colonies, while still maintaining high cloning accuracy. This increase in the length of sequence
homology is not detrimental to any standard cloning applications. For more details, please read the In-Fusion
Snap Assembly Multiple-Insert Cloning Protocol-At-A-Glance.

2. The 3’ portion of each primer should:

• be gene-specific.
• be between 18–25 bases in length and have a GC-content between 40–60%.
• have a melting temperature (Tm) between 58–65°C. The Tm difference between the forward and reverse
primers should be ≤4°C, or you will not get good amplification.
NOTE: The Tm should be calculated based upon the 3’ (gene-specific) end of the primer, and NOT the
entire primer. If the calculated Tm is too low, increase the length of the gene-specific portion of the primer
until you reach a Tm of between 58–65°C.
• not contain identical runs of nucleotides. The last five nucleotides at the 3’ end of each primer should
contain no more than two guanines (G) or cytosines (C).
3. Avoid complementarity within each primer to prevent hairpin structures, and between primer pairs to avoid
primer dimers.
4. You can perform a BLAST search to determine if the 3’ portion of each primer is unique and specific (at
www.ncbi.nlm.nih.gov/BLAST/).
5. We provide an online tool that simplifies In-Fusion PCR primer design for all possible In-Fusion Cloning
reactions:
takarabio.com/in-fusion-tools
6. We generally use desalted oligonucleotide primers in PCR reactions. However, primer quality can depend on
the vendor and varies from lot to lot. If your primer quality is particularly poor (i.e., has many premature
termination products), or your primers are longer than 45 nucleotides, they may need to be PAGE purified;
however, we usually find this is unnecessary.

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Figure 2. Universal primer design for In-Fusion technology. Successful insertion of a PCR fragment requires that the PCR insert shares
15 bases of homology with each end of the linearized vector. This sequence homology is added to the insert through the PCR primers. For
vectors with sticky ends, bases complementary to 5’ overhangs are included in the primer sequence; bases in the 3’ overhangs are not.

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Figure 3. Examples of primers designed for In-Fusion cloning. The above figure shows examples of primers designed with recognition
sites for restriction enzymes that generate: 5’ overhangs (Top), blunt ends (Middle), and 3’ overhangs (Bottom). The primer sequences are
shown in bold. The Xs represent bases corresponding to the gene or sequence of interest. Additional nucleotides (indicated with a black
box) have been added to each primer in order to reconstruct the restriction sites. They are not part of the 15 bases of sequence homology.

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Appendix B. pUC19 Linearized Vector Information

Figure 4. pUC19 Linearized Vector map and multiple cloning sites (MCS). pUC19 is a commonly used, high copy number cloning
vector. This linearized version was generated by PCR, and contains the blunt ends shown in the MCS sequence above. The vector encodes
the N-terminal fragment of β-galactosidase (lacZα), which allows for blue/white colony screening (i.e., α-complementation), as well as a
pUC origin of replication and an ampicillin resistance gene that allow propagation and selection in E. coli.

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