The AccuRapid™ Cloning Kit, developed by Bioneer, is engineered
to provide a streamlined and efficient solution for molecular cloning. Its
primary design focuses on the accurate and rapid insertion of PCR-amplified DNA
fragments, referred to as inserts, into a linearized vector DNA. A key
characteristic of this system is its capacity to facilitate the directional
cloning of one to three distinct insert fragments simultaneously. This
capability allows for more complex molecular constructions in a single
reaction, potentially accelerating research workflows.
The methodology
employed by the AccuRapid™ Cloning Kit circumvents
several steps common to traditional cloning protocols, notably the requirement
for restriction enzyme digestion of the PCR insert and
subsequent enzymatic ligation. By eliminating these steps for the insert, the
kit aims to reduce the overall time and complexity associated with gene
cloning. The product literature emphasizes that the kit is designed for
user-friendliness and high efficiency, catering to researchers who require both
speed and precision in their cloning experiments. The name chosen for the
product, "AccuRapid™," appears to
strategically reflect these core attributes of accuracy and speed, which are
consistently highlighted in the product descriptions. This nomenclature itself
serves as an immediate communication of the kit's primary value propositions to
potential users in the field of molecular biology.
The official
designation for this product is the AccuRapid™ Cloning
Kit. To accommodate varying experimental scales and laboratory needs,
Bioneer offers the kit in several sizes, each identified by a unique product
code:
● K-7110: Provides reagents for 10 cloning reactions.
● K-7120: Provides reagents for 20 cloning reactions, typically packaged as two 10-reaction sets.
● K-7130: Provides reagents for 50 cloning reactions, typically packaged as five 10-reaction sets.
The
availability of these different kit sizes suggests a flexible approach to
market needs, allowing researchers to select a quantity appropriate for
occasional use, pilot studies, or higher-throughput
cloning projects. This tiered offering can cater to individual labs with
limited cloning requirements as well as core facilities or research groups
performing cloning more extensively.
The AccuRapid™ Cloning Kit operates based on a
recombination-based cloning technology. This approach fundamentally differs
from conventional methods that depend on the specificities of restriction
endonucleases for generating compatible DNA ends and DNA ligases for their
subsequent covalent joining. Instead, the AccuRapid™
system utilizes an enzymatic process that recognizes and joins DNA fragments
based on sequence homology at their termini. This mechanism inherently bypasses
some of the common limitations associated with traditional cloning, such as the
reliance on the presence and suitability of restriction enzyme recognition
sites flanking the DNA fragment of interest, the potential for incomplete DNA
digestion by restriction enzymes, and the often variable
efficiency of the ligation step. The streamlined workflow offered by this
recombination-based approach contributes significantly to the "rapid"
characteristic of the kit.
Central to the
kit's mechanism is the utilization of short, 18 to 21 base pair (bp)
complementary DNA sequences, often referred to as homology arms. These
sequences must be present at the ends of the PCR-amplified insert(s) and the
corresponding ends of the linearized vector. The proprietary enzyme mix within
the kit is engineered to recognize these homologous regions and mediate the
annealing and subsequent joining of the DNA fragments.
These critical
18-21 bp homology sequences are incorporated into the insert
DNA during the PCR amplification step. This is achieved by designing PCR
primers that include these specific vector-complementary sequences as 5'
overhangs, in addition to the template-specific sequence required for
amplification of the desired insert. The defined length of these overhangs
(18-21 bp) is likely an optimized parameter for the enzyme system, balancing
the need for specific annealing with the efficiency of the recombination
process. The fidelity of these sequences is paramount, as the entire cloning
strategy hinges on their correct base-pairing.
A significant
advantage of the AccuRapid™ Cloning Kit is its
inherent capability for directional cloning, ensuring that the insert DNA is
integrated into the vector in a predetermined orientation. This is achieved
through the strategic design of the 18-21 bp complementary sequences on the
forward and reverse PCR primers used to amplify the insert.
Specifically, the
5' overhang of the forward primer is designed to be complementary to one end of
the linearized vector, while the 5' overhang of the reverse primer is designed
to be complementary to the other end of the linearized vector. Because these two
vector end sequences are distinct, the insert can only be stably incorporated
in one orientation, dictated by the specific homologies introduced by the
primers. This directional control is crucial for many downstream applications,
particularly in gene expression studies where the orientation of the coding
sequence relative to regulatory elements like promoters and terminators is
critical for function.
The AccuRapid™ Cloning Kit extends its utility beyond single
insert cloning by enabling the simultaneous assembly of multiple DNA fragments
(explicitly stated as 1 to 3 inserts) into a linearized vector in a specific,
predefined order. This multi-fragment assembly is also mediated by the 18-21 bp
complementary sequences.
For a
multi-fragment assembly (e.g., two inserts, I1 and I2, into a vector V), the
PCR primers are designed as follows:
● Insert I1: Forward primer has an overhang complementary to one end of vector V. Reverse primer has an overhang complementary to the 5' end of insert I2.
● Insert I2: Forward primer has an overhang complementary to the 3' end of insert I1. Reverse primer has an overhang complementary to the other end of vector V.
The
enzyme mix then facilitates the ordered assembly (V-I1-I2-V) based on these
specific homologous overlaps. This capability significantly enhances the kit's
versatility, allowing for the construction of more complex genetic constructs,
such as operons, fusion proteins from multiple domains, or synthetic pathways,
in a single cloning reaction. This is a notable advancement over methods
requiring sequential cloning steps for such assemblies.
The underlying
principle of joining DNA fragments via short homologous overlaps, mediated by
an enzyme mix, bears conceptual resemblance to established recombination-based
cloning techniques such as Gibson Assembly® or Sequence and Ligation
Independent Cloning (SLIC). User feedback referenced in the product flyer,
which mentions the convenience of the AccuRapid™ kit
compared to performing SLIC with individually purchased enzymes, further
supports this similarity. The AccuRapid™ kit likely
provides a proprietary, pre-optimized, and convenient formulation of enzymes
that execute these biochemical steps, offering researchers a ready-to-use
system. The "Enzyme Mix" is therefore the central, enabling component
of this technology. However, the success of this method is profoundly dependent
on the meticulous design of the PCR primers and the quality of the PCR
amplification process that generates the inserts with the correct terminal
sequences. Any errors introduced during primer synthesis or PCR, such as
incorrect overhang sequences or PCR-induced mutations within these overhangs,
could severely compromise or entirely prevent successful cloning.
The AccuRapid™ Cloning Kit is supplied with essential reagents designed to perform the cloning reaction and validate its efficacy. The components included in each kit size (K-7110, K-7120, K-7130).
This is the
central functional component of the kit. It is a proprietary blend of enzymes
presumed to contain the necessary activities (which might include exonucleases,
DNA polymerases, DNA ligases, or specific recombinases) to recognize the 18-21
bp homologous overhangs on the DNA fragments and mediate their annealing and
covalent joining. The precise composition and concentrations of the enzymes
within this mix are not disclosed by the manufacturer, which is a common
practice for commercial biotechnology kits. This "black box" nature
means users rely on Bioneer's formulation and
optimization. Recent product literature indicates that the volume of this
enzyme mix has been reduced, while the permissible DNA input volume has been
increased, with the aim of enhancing cloning efficiency, particularly when
assembling multiple insert fragments.
The kit includes
a 2 kb control vector, designated pBHA, supplied at a
concentration of 25 ng/µl. This vector is provided in a linearized form, ready
for use in a control cloning reaction. Its primary purpose is to allow users to
verify the functionality of the kit's reagents and the overall cloning protocol.
Successful cloning of the control insert into this
control vector provides confidence in the kit's performance and can aid in
troubleshooting if users encounter issues with their experimental samples.
A 750 bp control
DNA fragment is also provided, at a concentration of 50 ng/µl. This fragment is
designed as a PCR product with appropriate ends for cloning into the pBHA Control Vector. When used in conjunction with the
control vector and the AccuRapid™
Enzyme Mix, it serves as a positive control for the entire cloning procedure.
The successful outcome of this control reaction (i.e., efficient cloning of the
750 bp insert into the 2 kb vector) confirms that all kit components are active
and that the reaction conditions are appropriate.
The inclusion of
both a control vector and a control insert is a
valuable feature. It provides a robust system for users to validate the kit's
performance independently of their own experimental DNA fragments. Should a
researcher's specific cloning experiment yield suboptimal results, a successful
control reaction would suggest that the issue likely lies with the user's
specific vector preparation (e.g., incomplete linearization), insert PCR (e.g.,
poor yield, incorrect overhangs), or primer design, rather than a problem with
the AccuRapid™ kit reagents themselves. This
systematic troubleshooting capability is an indicator of thoughtful kit design.
|
Component |
Provided Concentration/Details |
Primary Function in the Kit |
|
AccuRapid™
Enzyme Mix |
Not specified |
Provides enzymatic
activities for recognizing and joining DNA fragments with complementary ends
(recombination/ligation). |
|
2 kb pBHA
Control Vector |
25 ng/µl, linearized |
Serves as a positive control
vector for experimental validation and troubleshooting. |
|
750 bp Control Insert |
50 ng/µl, PCR product |
Serves as a positive control
insert for cloning into the pBHA Control Vector,
validating kit performance. |
The experimental
procedure for the AccuRapid™ Cloning Kit, as outlined
in the Quick Manual, involves several key stages from DNA fragment preparation
to the assembly of the final cloning reaction.
The initial step
requires the preparation of a suitable linearized vector. This can be achieved
by either PCR amplification of the entire vector backbone using primers that
generate the desired ends, or, more commonly, through digestion of a circular
plasmid vector with one or more restriction enzymes. The product literature
strongly emphasizes the critical importance of using a "completely
linearized vector" to achieve high cloning efficiency. Residual undigested
or partially digested circular vector can transform host cells with high
efficiency, leading to a significant background of colonies that do not contain
the desired insert. This, in turn, reduces the apparent efficiency of the
cloning reaction. Verification of complete linearization, for instance, by
agarose gel electrophoresis (potentially comparing digested vector with undigested
control or confirming the shift to a single linear band of the correct size),
is implicitly recommended to ensure optimal results.
The DNA insert(s)
to be cloned are typically generated by PCR amplification. As detailed in
Section 5, the PCR primers used for this amplification must be designed with
specific 5' overhangs, 18 to 21 base pairs in length. These overhangs must
contain sequences that are complementary to the ends of the linearized vector
where the insert is intended to be integrated. Following PCR amplification, it
is recommended to purify the PCR product(s). This purification step, commonly
performed using a commercial PCR/gel purification kit, is crucial for removing
PCR contaminants such as residual primers, primer-dimers, dNTPs, polymerase,
and buffer components, any of which could potentially inhibit the subsequent
enzymatic cloning reaction. The quality and purity of the PCR-generated insert,
including the presence of correct and intact overhangs, are paramount for
successful cloning.
The cloning
reaction itself is assembled in a microcentrifuge tube by combining the
prepared linearized vector, the purified PCR product(s), the AccuRapid™ Enzyme
Mix, and nuclease-free distilled water to achieve a final reaction volume of 10
µl. The Quick Manual provides specific recommendations for the amounts of DNA
to be used:
● Linearized Vector: 25-50 ng (typically in 1 µl volume).
● Purified PCR Product(s): A total of 70-150 ng. The volume for PCR products can vary but the total DNA input (vector + insert(s)) should not exceed 5 µl.
○ For cloning multiple fragments, the total amount of PCR product (70-150 ng) is maintained, but this amount is distributed among the individual fragments. For example, if using 5 µl total for inserts: for 1 fragment, 5 µl; for 2 fragments, 2.5 µl of each; for 3 fragments, approximately 1.7 µl of each.
It
is noted that recent updates to the kit have involved an increase in the
permissible DNA input volume, which is intended to improve cloning efficiency,
especially when dealing with multiple inserts. This suggests an ongoing effort
by the manufacturer to refine reaction stoichiometry for more demanding
applications.
Once the reaction
components are mixed, the tube should be gently tapped and briefly centrifuged
to collect the contents at the bottom. The cloning reaction is then incubated
for 30 minutes at 50°C. This
specific combination of time and temperature is presumably optimized for the
enzymatic activities within the AccuRapid™ Enzyme Mix. The 50°C incubation temperature may
facilitate DNA strand annealing or exchange, or
represent the thermal optimum for one or more key enzymes in the mix. The short
30-minute duration is a key feature contributing to the "Rapid"
aspect of the kit's name and a significant practical advantage.
Following the
30-minute incubation at 50°C, the reaction mixture should be immediately placed
on ice if transformation is to be performed shortly thereafter,
or stored at -20°C for longer-term storage prior to transformation. The
protocol then directs the user to add the reaction mixture (presumably the
entire 10 µl volume, though this should be confirmed with the full user guide)
to competent E. coli cells for
transformation. Notably, the Quick Manual protocol does not explicitly mention
a heat inactivation step to denature the enzymes in the AccuRapid™
Mix prior to transformation. While some enzyme mixes benefit from such a step
to prevent interference with transformation or plasmid stability, its absence
here might imply that the enzymes are not detrimental, their activity
sufficiently ceases at lower temperatures, or this detail is covered in the
comprehensive User Guide.
|
Component |
Recommended Amount/Volume |
Notes |
|
Linearized Vector |
25-50 ng (typically 1 µl) |
Must be completely
linearized. |
|
Purified PCR Product(s) |
70-150 ng total (variable
volume, up to 5 µl max for DNA) |
For multiple fragments,
divide volume (e.g., 2 fragments: 2.5 µl each; 3 fragments: ~1.7 µl each). |
|
AccuRapid™ Enzyme Mix |
4 µl |
|
|
Distilled Water |
Variable (adjust to reach
total volume of 10 µl) |
|
|
Total Volume |
10 µl |
|
The explicit
instructions for upstream sample preparation, such as the purification of PCR
products and the critical need for a "completely linearized vector",
underscore their importance. These prerequisites suggest that the AccuRapid™ Enzyme Mix may be sensitive to common
contaminants found in unpurified PCR reactions or that the presence of even
small amounts of residual circular vector DNA can significantly skew results by
outcompeting the desired recombination products during transformation.
Therefore, meticulous attention to these pre-reaction steps is as vital to
success as the cloning reaction itself.
The success of
cloning experiments using the AccuRapid™ Cloning Kit
is fundamentally dependent on the correct design of PCR primers used to amplify
the insert DNA. These primers are responsible for introducing the necessary
terminal sequences that the kit's enzymes recognize for recombination.
The core
principle of primer design for this system is the incorporation of an 18 to 21
base pair sequence at the 5' end of both the forward and reverse primers. These
5' extensions, or overhangs, are not complementary to the target template DNA
being amplified; instead, they must be designed to be complementary to the
sequences at the precise ends of the linearized vector into which the insert
will be cloned. During PCR, these overhangs are incorporated into the
amplicons, furnishing them with the "homology arms" required for the AccuRapid™ cloning reaction. The accuracy of these 18-21 bp
sequences is paramount, as they dictate the specificity of the subsequent
recombination event.
To achieve
directional cloning, which is often essential for functional gene expression,
the 5' overhangs on the forward and reverse primers must be designed with
distinct sequences corresponding to the two different ends of the linearized
vector. Specifically:
● The 18-21 bp overhang on the 5' end of the forward primer should be complementary to the sequence at one chosen end of the linearized vector (e.g., if the vector is linearized at site A and site B, this overhang matches site A).
● The 18-21 bp overhang on the 5' end of the reverse primer should be complementary to the sequence at the other end of the linearized vector (e.g., matching site B).
This
differential design ensures that the PCR product can only be integrated into
the vector in a single, predetermined orientation, as stable recombination will
only occur when both ends of the insert correctly anneal to their complementary
sequences on the vector.
The
AccuRapid™ kit's capability to clone multiple DNA
fragments (1 to 3 are specified) simultaneously into a vector relies on an
extension of this primer design strategy. Each fragment to be assembled must be
amplified by PCR using primers that generate the appropriate 18-21 bp
homologous ends for ordered assembly.
For
example, to clone two inserts (Insert A and Insert B) between two vector ends
(Vector End 1 and Vector End 2) in the order
VectorEnd1-InsertA-InsertB-VectorEnd2:
● Insert A Primers:
○ Forward Primer: 5' overhang complementary to Vector End 1.
○ Reverse Primer: 5' overhang complementary to the 5' sequence of Insert B.
● Insert B Primers:
○ Forward Primer: 5' overhang complementary to the 3' sequence of Insert A (i.e., the sequence introduced by Insert A's reverse primer overhang).
○ Reverse Primer: 5' overhang complementary to Vector End 2.
This
creates a chain of homologous overlaps that allows the enzyme mix to assemble
the fragments in the desired sequence and orientation relative to each other
and to the vector. While this offers considerable flexibility for constructing
complex plasmids, it also significantly increases the complexity of primer
design. Each of the 18-21 bp overhang sequences must be carefully planned to
ensure unique and correct pairing, avoiding unintended interactions or
mis-assemblies. The potential for error in designing multiple, compatible
overhangs rises with the number of fragments.
While the AccuRapid™ cloning mechanism relies solely on sequence
homology and does not require restriction enzyme recognition sites within the
18–21 bp overhangs, the inclusion of these sites—whether complete or
partial—can have functional implications that merit careful consideration. If
a complete restriction enzyme recognition site (e.g., NdeI,
XhoI) is incorporated within an overhang, the site
can be fully reconstituted in the final construct. This allows for subsequent
enzymatic digestion or cloning verification and can be advantageous for
downstream applications requiring site-specific excision, screening, or modular
assembly. Conversely, if only a partial restriction site is included
in the overhang, the resulting site will be non-functional and thus uncleavable
by the corresponding enzyme. However, residual sequences from this partial site
will persist in the final product. These leftover nucleotides can interfere
with reading frames, introduce unintended codons, or affect sequence-based
annotations. If no restriction enzyme site is included, users should be
aware that cloning efficiency might decrease. This is particularly true if
overhangs are short, non-specific, or lack the GC content or sequence
complexity necessary for stable and specific annealing. Consequently, excluding
restriction sites is generally not recommended unless validated by prior
optimization or experience. Therefore, users should
decide whether to include full, partial, or no restriction site sequences in
the overhangs based on their specific project goals. For high-fidelity or
expression-sensitive applications, it is often advisable to use clean,
non-disruptive overhangs or to incorporate full restriction sites only if their
preservation offers a distinct functional advantage.This
simplifies primer design, as users are not constrained by the need to find or
incorporate specific restriction sites into these overhangs, unless such sites
are desired for other downstream manipulations of the cloned construct.
The available
product information, including the Quick Manual and the product webpage
sections on primer design, provides foundational guidelines. However, these
sources also indicate that more comprehensive information, potentially
including detailed examples, troubleshooting for primer design, and
considerations for complex templates, can be found in the main User Guide for
the AccuRapid™ Cloning Kit (referenced as
UG_AccuRapid_Cloning_Kit.pdf). Users are advised to consult this detailed
manual for exhaustive guidance.
The generation of
inserts with precise terminal sequences via PCR implies a strong, albeit
unstated, recommendation for using a high-fidelity DNA polymerase for the
amplification step. Standard DNA polymerases lacking proofreading activity can
introduce errors (mutations) during PCR. If such errors occur within the
crucial 18-21 bp overhang sequences, they could abolish or significantly reduce
the homology to the vector or adjacent fragments, leading to failed
or inefficient cloning. Therefore, employing a high-fidelity polymerase is a
critical best practice to ensure the integrity of these sequences.
The AccuRapid™ Cloning Kit is marketed with several performance
claims and user benefits, positioning it as an attractive tool for molecular
cloning workflows.
A standout
feature, consistently emphasized in the product literature, is the remarkably
short cloning reaction time: just 30 minutes at 50°C. This rapid reaction
significantly reduces the overall time required to move from prepared DNA
fragments (linearized vector and PCR insert) to a reaction mixture ready for
transformation. Compared to traditional cloning protocols, which often involve
overnight ligations or multiple sequential enzymatic steps spanning several
hours, this 30-minute protocol offers a substantial time saving, accelerating
experimental progress.
The kit is
described as highly efficient. Particular emphasis is
placed on its efficiency in scenarios involving the insertion of multiple DNA
fragments. Product information highlights that recent modifications to the kit formulation, such as a reduction in the enzyme mix
volume and an increase in the allowable DNA input volume, were specifically
implemented to enhance the efficiency of multi-fragment cloning. While the term
"high cloning efficiency" is used, the publicly available product
information and flyer do not provide specific quantitative performance data,
such as the percentage of positive clones typically obtained for single versus
multiple inserts, or across a range of insert sizes. Such detailed metrics are
often found in comprehensive user manuals or dedicated application notes. The
documentation does, however, stress that achieving this high efficiency is
critically dependent on using a "completely linearized vector," as
this minimizes background colonies arising from the parental vector carryover.
The AccuRapid™ Cloning Kit facilitates precise and directional
cloning of the insert(s) into the vector. As discussed previously, the
directionality is governed by the unique 18-21 bp homologous sequences designed
by the user at the 5' ends of the PCR primers. This ensures that the insert is
integrated in the correct orientation relative to vector elements, which is
vital for the functional expression of cloned genes or regulatory sequences.
The kit is
presented as a convenient, easy-to-use system. User reviews, cited in the
product flyer, specifically highlight this convenience, particularly when
comparing the AccuRapid™ kit to methods like SLIC
(Sequence and Ligation Independent Cloning) where researchers might procure and
optimize individual enzymes and buffers themselves. The pre-mixed, optimized
enzyme formulation and straightforward protocol contribute to this ease of use,
reducing hands-on time and the potential for errors associated with preparing
multiple reagents from scratch. This convenience is a trade-off for the lack of
detailed information about the enzyme mix composition, but for many users, the
reliability and time savings of an optimized commercial kit outweigh the desire
for component-level control.
The AccuRapid™ Cloning Kit is promoted as being available at an
"economical price" when compared to competitor offerings. This claim
is supported by at least one user review mentioned in the product flyer, which explicitly
states that the Bioneer product is cheaper than a comparable cloning kit from
"company T" (a common reference to Takara Bio, a well-known supplier
of cloning reagents), leading to high user satisfaction from a cost
perspective. While "economical" is relative, this suggests a
competitive pricing strategy.
User testimonials
included in the promotional materials suggest that the kit delivers reliable
and consistent cloning results. These reviews indicate performance that is
comparable to, or in some cases better than, alternative products or more
laborious traditional methods. For instance, one review noted "no
significant difference from using individually purchased enzymes"
(implying it performs as effectively as a more complex, do-it-yourself
approach), while another reported "better efficiency than company T's
kit". Such anecdotal evidence, while not a substitute for direct,
peer-reviewed comparative studies, provides an indication of user satisfaction
and the kit's general reliability in practical research settings. However, for
rigorous evaluation, especially when comparing against established
alternatives, researchers would ideally seek direct, quantitative comparative
data, which is not extensively provided in the summarized materials.
The design and
features of the AccuRapid™ Cloning Kit make it
suitable for a range of common molecular cloning tasks, particularly those
centered around PCR-generated DNA fragments.
The foremost
application of the AccuRapid™ Cloning Kit is the
cloning of DNA inserts that have been generated through PCR amplification. The
entire methodology, from primer design to the enzymatic reaction, is optimized
for DNA fragments produced by PCR, which are readily modified to include the
required 18-21 bp terminal homologies. This focus makes the kit highly relevant
for numerous standard molecular biology workflows, such as isolating genes from
genomic or cDNA, amplifying mutated gene variants produced by site-directed
mutagenesis, or generating DNA fragments for protein domain swapping
experiments.
The kit
streamlines the overall cloning process by facilitating the rapid preparation
of inserts. Because the cloning mechanism relies on homologous sequences added
during PCR, it obviates the need for subsequent restriction enzyme digestion of
the PCR product to create compatible ends. This not only saves time but also
offers an advantage when suitable restriction sites are absent or
inconveniently located within or around the insert.
A key utility of
the kit is its ability to ensure the accurate directional cloning of inserts
into the vector. This is critical for applications where the orientation of the
cloned fragment is important, most notably in the construction of expression
vectors where a gene must be placed downstream of a promoter in the correct
reading frame and orientation.
The AccuRapid™ Cloning Kit offers versatility by supporting the
cloning of both single DNA inserts and the assembly of multiple fragments (up
to three are explicitly mentioned) in a single, ordered reaction. This
capability extends its use from simple subcloning tasks to more moderately
complex genetic engineering projects. For example, it can be used to assemble a
promoter, a gene of interest, and a terminator sequence into an expression
cassette, or to construct a fusion protein from two or three separately
amplified domains. This addresses a common need for creating multi-component
genetic systems without resorting to multiple rounds of sequential cloning.
Beyond specific
examples, the product flyer suggests that the AccuRapid™
Cloning Kit is suitable for a broad variety of molecular biology applications
where gene cloning is a fundamental step. The product webpage also highlights
its "versatile cloning design," which allows for "easy
manipulation of the vector into the desired form", indicating its
adaptability to diverse research objectives within the field.
The AccuRapid™ Cloning Kit is offered by Bioneer in three
different package sizes to suit varying laboratory demands. Each size is
designated by a specific product code for ease of ordering:
● K-7110: This kit provides sufficient reagents for 10 individual cloning reactions.
● K-7120: This kit provides reagents for a total of 20 cloning reactions. It is packaged as two separate sets, each containing reagents for 10 reactions (10 reactions x 2).
● K-7130: This is the largest available size, providing reagents for 50 cloning reactions. It is packaged as five separate sets, each for 10 reactions (10 reactions x 5).
The
modular packaging of the larger kit sizes (K-7120 and K-7130) as multiples of
10-reaction units is a practical consideration. This approach may be intended
to enhance reagent stability by allowing users to open and utilize one
10-reaction aliquot of enzymes and other sensitive components at a time. This
minimizes the number of freeze-thaw cycles or prolonged exposure to ambient
conditions for the remaining aliquots, thereby helping to maintain the
integrity and optimal performance of the reagents over the shelf life of the
kit.
|
Product Code |
Kit Size (Number of Reactions) |
Packaging Detail |
|
K-7110 |
10 reactions |
N/A |
|
K-7120 |
20 reactions |
10 reactions x 2 |
|
K-7130 |
50 reactions |
10 reactions x 5 |
Successful
application of the AccuRapid™ Cloning Kit relies on
attention to several critical technical details, and users should be aware of
where to find comprehensive information.
The product
documentation consistently and strongly emphasizes that the use of a
"perfectly" or "completely" linearized vector is paramount
for achieving high cloning efficiency with the AccuRapid™
kit. If the vector preparation contains a significant amount of undigested or
nicked circular plasmid, these forms will transform E. coli with much higher efficiency than the desired recombinant
constructs. This results in a high background of colonies that only contain the
empty vector, thereby drastically reducing the apparent efficiency of
successful insert cloning and making the identification of positive clones more
laborious. It is therefore implicitly recommended that users verify the
completeness of vector linearization, for example, by agarose gel electrophoresis
analysis, potentially including a control lane with undigested plasmid to
confirm a shift in mobility and the absence of supercoiled or circular forms in
the digested sample. This upstream step, while not part of the kit's direct
reaction, is a major determinant of its effective performance.
After the
30-minute cloning reaction at 50°C is complete, the protocol advises that the
reaction mixture should be stored either on ice for short-term storage (if
proceeding to transformation relatively soon) or at -20°C for longer-term
storage before transformation. This practice is important to maintain the
stability of the newly assembled DNA constructs and to halt or significantly
slow down any residual enzymatic activity that might, over time, lead to
degradation of the products or other undesirable modifications prior to their
introduction into competent host cells.
While the product
webpage and associated flyer provide a good overview of the AccuRapid™
Cloning Kit, several specific technical parameters that are often crucial for
detailed experimental planning and troubleshooting are not explicitly defined
in these summarized materials:
● Insert DNA Size Limitations (Minimum/Maximum): Although a 750 bp control insert is provided and used as an example, and the kit is stated to clone 1-3 insert pieces, the acceptable working range for insert DNA size (e.g., lower limit of ~100 bp to an upper limit of several kilobases) is not specified in the readily accessible documents.
● Detailed Vector Compatibility Requirements (Beyond Linearization): Beyond the fundamental requirement that the vector must be linearizable and possess ends to which complementary primer overhangs can be designed, other potential vector compatibility issues (such as limitations on overall vector size, or the presence of specific sequences within the vector that might interfere with the cloning reaction) are not detailed.
● Use of Restriction Fragments as Inserts: The kit is heavily marketed and its protocol designed for the use of PCR-amplified inserts, which incorporate the necessary overhangs via primers. Whether pre-existing DNA fragments generated by restriction enzyme digestion (and subsequently modified to have the correct 18-21 bp terminal homologies, perhaps through ligation of adaptors or a fill-in/exonuclease treatment strategy) can be efficiently used as inserts is not explicitly addressed. The general emphasis is on avoiding restriction enzyme treatment of the insert itself.
● Kit Shelf Life and Recommended Storage Conditions: Specific information regarding the shelf life of the unopened kit or its individual components (particularly the AccuRapid™ Enzyme Mix), and precise long-term storage conditions (e.g., -20°C versus -80°C for optimal enzyme stability), are not mentioned in the summarized product page or flyer.
The
absence of such explicit specifications in easily accessible marketing
materials can present a challenge for researchers attempting to thoroughly
evaluate the kit against alternatives or for specific, demanding applications.
While this information is likely available, its omission from primary
promotional documents necessitates additional steps from the user to obtain it.
For researchers
requiring more detailed technical specifications or guidance beyond what is
available on the product webpage and flyer, Bioneer directs users to two
primary resources:
1. The comprehensive User Guide for the AccuRapid™ Cloning Kit. This document is linked on the product page (filename: UG_AccuRapid_Cloning_Kit.pdf 1) and is expected to contain more exhaustive information on all aspects of the kit, including detailed protocols, troubleshooting, and potentially the missing specifications noted above. References within the Quick Manual and primer design section point to this User Guide as the definitive source for in-depth information.
2. Bioneer's Technical Support: Direct contact with Bioneer's customer and technical support team is encouraged for specific queries. Contact information provided includes a main phone number (1588-9788) and an email address for product inquiries and technical support (science_support@bioneer.co.kr).
It was noted
during the information gathering process that one of the hyperlinks on the
product page, labeled "실험 방법" (Experimental Method) and pointing to an image file
(https://www.bioneer.co.kr/images/products/AccuRapid_Cloning_Kit_TC02.jpg), was
inaccessible. This image might have provided a visual summary of the protocol
or other pertinent experimental details that could not be assessed.
The AccuRapid™ Cloning Kit from Bioneer presents itself as a
modern, efficient, and user-friendly solution for the cloning of PCR-amplified
DNA fragments. Its core technology, based on the recombination of DNA fragments
sharing 18-21 base pair homologous ends, offers significant advantages in terms
of speed and simplicity over traditional restriction enzyme and
ligase-dependent cloning methods.
The kit's most
prominent benefits include a remarkably rapid 30-minute cloning reaction, the
capability for accurate directional cloning, and the efficient assembly of
single or multiple (up to three) insert fragments simultaneously. User feedback
highlighted in promotional materials suggests a high degree of convenience and
cost-effectiveness, particularly when compared to some competitor kits or more
laborious "do-it-yourself" recombination cloning approaches. These
attributes position the AccuRapid™ kit as a
competitive option in the molecular cloning market.
The AccuRapid™ Cloning Kit is well-suited for a variety of
common molecular biology applications that require the cloning of PCR products.
This includes routine subcloning, construction of expression vectors, and the
assembly of moderately complex genetic constructs such as multi-domain fusion
proteins or simple synthetic pathways. Its ability to handle multiple inserts
in an ordered fashion is a particularly valuable feature for researchers
engaged in synthetic biology or multi-gene expression studies.
To achieve
optimal results with the AccuRapid™ Cloning Kit,
users must pay careful attention to several critical upstream factors. These
include meticulous primer design to ensure the correct 18-21 bp overhang
sequences for homology, the preparation of high-quality, thoroughly purified
PCR products, and, crucially, ensuring the complete linearization of the vector
DNA to minimize background from non-recombinant plasmids. Success with the kit
is highly dependent on the user's proficiency in these preparative steps.
While the product
webpage and flyer provide a useful introduction, potential users seeking
comprehensive technical details—particularly concerning insert size
limitations, exhaustive vector compatibility parameters, and definitive shelf life information—are strongly encouraged to consult
the full User Guide (UG_AccuRapid_Cloning_Kit.pdf). For any remaining
ambiguities or highly specific application queries, direct contact with Bioneer's technical support services is recommended. A
thorough understanding of these parameters will enable researchers to make
informed decisions about the kit's suitability for their specific experimental
needs and to maximize their chances of successful cloning outcomes. The AccuRapid™ Cloning Kit appears to balance its evident
strengths in speed and multi-fragment assembly with the need for users to
diligently prepare their starting materials and seek out comprehensive
documentation for advanced applications.