TA cloning
represents a widely adopted molecular biology technique for the direct ligation
of Polymerase Chain Reaction (PCR) products into a plasmid vector. This method
leverages the terminal transferase activity inherent to certain DNA
polymerases, most notably Taq DNA
polymerase. During PCR amplification, these polymerases preferentially add a
single deoxyadenosine (A) nucleotide to the 3'-ends of the double-stranded PCR
fragments, creating a 3'-A overhang. Consequently, these A-tailed PCR products
can be efficiently ligated into a specialized cloning vector that has been
linearized and engineered to possess complementary single 3'-deoxythymidine (T)
overhangs at its ends.
A significant
advantage of TA cloning is its simplicity, as it circumvents the need for
restriction enzyme digestion of the PCR product or the laborious incorporation
of specific restriction enzyme recognition sites into the PCR primers, which is
often a prerequisite for traditional cloning strategies. This characteristic
makes TA cloning particularly useful for rapidly cloning diverse PCR products,
including those from genomic DNA or cDNA, where convenient restriction sites
may not be available or when the sequence is unknown.
The dependence on
polymerase-added 3'-A overhangs underscores the critical importance of
selecting an appropriate DNA polymerase for generating the insert DNA. While
non-proofreading polymerases like Taq
are ideal due to their natural A-tailing activity, high-fidelity proofreading
polymerases possess a 3'→5' exonuclease activity. This exonuclease function,
while crucial for correcting errors during DNA synthesis, will remove any 3'-A
overhangs, rendering the PCR product blunt-ended and thus incompatible with
direct TA cloning. If a proofreading polymerase is employed for applications
demanding high sequence accuracy, a subsequent enzymatic step, known as
A-tailing (typically involving incubation with a non-proofreading polymerase
and dATP), becomes necessary to add the required 3'-A
overhangs. This additional step can introduce variability and potentially
reduce overall cloning efficiency if not optimized.
Furthermore, the
inherent nature of TA cloning, which relies on the single A/T overhang
complementarity, results in the insert ligating into the vector in either of
two possible orientations (forward or reverse). This is because the T-overhangs
on the vector are identical, and the A-overhangs on the insert are also
identical at both ends. Unlike methods employing two distinct restriction
enzymes or sequence-specific recombination (such as the AccuRapid™
Cloning Kit K-7110 5), standard TA cloning does not offer inherent
directionality. The orientation of the insert must therefore be determined
post-cloning, typically through restriction analysis or sequencing.
The Bioneer AccuRapid™ TA Cloning Kit, product code K-7170, is
specifically engineered for the rapid and convenient cloning of PCR products
amplified with Taq DNA polymerase, or
other polymerases that generate 3'-A overhangs. The kit facilitates the direct
insertion of these A-tailed PCR products into the supplied pBHA-T
vector, which is pre-linearized and features complementary 3'-T overhangs on
each of its ends.
According to the
manufacturer, this kit is intended for research purposes only and is not
validated or supplied for diagnostic applications. This disclaimer is a
standard convention for molecular biology reagents not subjected to the
rigorous validation required for clinical diagnostics.
The product name
"AccuRapid™ TA Cloning Kit" itself suggests
key performance attributes. The "Rapid" component is substantiated by
the notably short ligation reaction time of just 15 minutes. The
"Accu" part of the name, in the context of TA cloning, likely refers
to the efficiency and reliability of the ligation process, leading to a high
yield of desired recombinant clones, rather than the sequence fidelity of the
cloned insert. Sequence fidelity in PCR-based cloning is primarily determined
by the error rate of the DNA polymerase used for amplification, with Taq polymerase being known for a
relatively higher error rate compared to proofreading polymerases. Therefore,
the "accuracy" here likely pertains to the robustness and success
rate of the cloning reaction itself.
The fundamental
scientific principle underlying the AccuRapid™ TA
Cloning Kit is the enzymatic ligation of DNA fragments possessing compatible,
single-nucleotide overhangs. Specifically, the kit employs T4 DNA Ligase to
catalyze the formation of phosphodiester bonds between the 3'-hydroxyl group of
the terminal thymidine on the linearized pBHA-T
vector and the 5'-phosphate group of the terminal adenosine on the
PCR-amplified insert DNA. Similarly, it joins the
3'-hydroxyl of the insert's A-overhang to the 5'-phosphate of the vector's
T-overhang. This process effectively incorporates the PCR product into the
plasmid vector.
A key feature of
the AccuRapid™ TA Cloning Kit is the optimization of
this ligation reaction for speed and efficiency. The reaction is designed to
proceed to completion within 15 minutes when incubated at 25°C. This rapid
ligation is a significant improvement over traditional ligation protocols,
which often require several hours or overnight incubations, sometimes at lower
temperatures (e.g., 16°C or 4°C).
The success of
this ligation is critically dependent on several factors. Firstly, the PCR
insert must possess intact and efficiently generated 3'-A overhangs. This is
typically achieved by using a DNA polymerase with terminal transferase
activity, like Taq polymerase, and
ensuring a sufficient final extension step during PCR. Secondly, the pBHA-T vector must have well-defined and undamaged 3'-T overhangs. Degradation of these single-stranded overhangs on
either the insert or the vector, for instance, due to nuclease contamination,
excessive freeze-thaw cycles, or prolonged storage, can severely diminish the
number of compatible ends available for ligation, thereby reducing cloning
efficiency. The T4 DNA Ligase itself requires specific cofactors, primarily ATP
and Mg2+, which are provided in the AccuRapid™ 2X
Reaction Buffer, to function optimally.
The Bioneer AccuRapid™ TA Cloning Kit (K-7170, 20 reactions) is
supplied with the essential reagents required for the ligation step. The
components and their respective roles are detailed below:
Table 1: Components of the AccuRapid™
TA Cloning Kit (K-7170)
|
Component Name |
Concentration |
Volume (for K-7170) |
Primary Function |
|
pBHA-T vector |
25 ng/µl |
40 µl |
Linearized plasmid vector
with 3'-T overhangs; serves as the cloning backbone for accepting A-tailed
PCR inserts. |
|
Control Insert |
70 ng/µl |
20 µl |
A pre-prepared, A-tailed DNA
fragment of known size (tested range 500 bp – 2,000 bp); used as a positive
control for the ligation and transformation steps. |
|
AccuRapid™ 2X Reaction Buffer |
- |
100 µl |
Provides the optimal
chemical environment (pH, salts, Mg2+, ATP) for T4 DNA Ligase activity,
enabling efficient ligation. |
|
T4 DNA Ligase |
200 U/µl (Weiss units) |
20 µl |
Enzyme
that catalyzes the formation of
phosphodiester bonds between the vector and insert DNA, sealing the nicks to
form a circular recombinant plasmid. |
Note: One Weiss unit (200 U) of T4 DNA Ligase is defined as
the amount of enzyme required to ligate 90% of HindIII
fragments of 1 µg of lambda DNA in a total volume of
20 µl at 16°C for 30 minutes.
The provision of
a Control Insert is a valuable feature for users. If an experimental cloning
reaction fails or yields suboptimal results, performing the cloning procedure
with the Control Insert can help diagnose the source of the problem. Successful
cloning of the Control Insert would suggest that the kit components (vector,
ligase, buffer) and the downstream transformation and screening procedures are
functioning correctly, thereby pointing towards an issue with the user's
experimental PCR product (e.g., inadequate A-tailing, presence of inhibitors,
incorrect concentration, or degradation). Conversely, failure of the control
reaction would indicate a potential problem with one or more of the kit's core
components or the subsequent experimental steps.
The pBHA-T vector, supplied with the AccuRapid™
TA Cloning Kit, is specifically designed for TA cloning and incorporates
several features to facilitate the selection, screening, and analysis of
recombinant clones. While the exact size is not explicitly stated for pBHA-T, TA cloning vectors based on pUC
architecture (like pMD18-T 7) are typically around 2.7 kb. The pBHA vector system, in general, is utilized for various
molecular biology applications, including expression. The features of the pBHA-T variant are summarized in Table 2.
Table 2: Key Features of the pBHA-T
Vector
|
Feature |
Description/Function |
Implication for User |
|
lacZα gene |
Encodes the N-terminal
α-fragment (amino acids 1-146) of β-galactosidase. The cloning site is
located within this gene. |
Enables blue/white screening
for recombinants. Insertion of DNA disrupts lacZα, leading to white colonies in the presence of IPTG and
X-gal on an appropriate E. coli
host (e.g., DH5α) that provides ω-complementation. Blue colonies typically
contain non-recombinant vector. |
|
Ampicillin Resistance Gene |
Confers
resistance to the antibiotic
ampicillin. |
Allows for selection of
bacterial cells that have been successfully transformed with the plasmid
(either recombinant or non-recombinant) by plating on media containing
ampicillin. |
|
M13 Forward & Reverse
Primer Sites |
Standard sequencing primer
binding sites flanking the multiple cloning site (MCS) within the lacZα gene. |
Facilitates sequencing of
the cloned insert from both directions using universal M13 primers to verify
insert identity, orientation, and integrity. |
|
T7 Promoter & SP6
Promoter |
Promoter sequences also
flanking the MCS. |
Primarily allows for in vitro RNA transcription of the
cloned insert to generate RNA probes or transcripts. These sites can also
potentially be used for sequencing. |
|
BamHI Restriction Site(s) |
Restriction enzyme sites
flanking the cloned insert. |
Enables easy excision of the cloned insert from the pBHA-T
vector using a single BamHI digestion. This is
advantageous for subcloning the insert into other vectors compatible with BamHI. |
|
pUC ori |
High-copy-number origin of
replication derived from pUC plasmids. |
Ensures high yields of
plasmid DNA from bacterial cultures, facilitating downstream applications
that require larger quantities of plasmid. |
The combination
of M13, T7, and SP6 primer binding sites offers considerable flexibility for
the characterization of the cloned insert. While M13 primers are the standard
choice for Sanger sequencing of inserts in many cloning vectors, the T7 and SP6
sites provide alternative sequencing options and the capability for in vitro transcription, which can be
useful for generating RNA for functional studies or probe synthesis.
The inclusion of BamHI sites flanking the insert is a practical feature that
simplifies the process of moving the cloned fragment into a different
expression or analysis vector, provided the recipient vector has compatible BamHI cloning sites. This can save time and effort compared
to strategies requiring multiple or different restriction enzymes for
subcloning.
The quality and
characteristics of the PCR product used as the insert are paramount for
achieving successful TA cloning with the AccuRapid™
TA Cloning Kit. Several critical considerations must be addressed during insert
preparation:
1. Polymerase Selection: The DNA polymerase used for PCR amplification must be capable of adding a single deoxyadenosine (A) to the 3'-ends of the PCR products. Taq DNA polymerase is the standard choice for this purpose due to its inherent terminal transferase activity. Bioneer recommends its AccuPower® PCR PreMix (Cat. No. K-2012) or AccuPower® Taq PCR PreMix (Cat. No. K-2601) for generating suitable inserts. If a high-fidelity proofreading polymerase (which typically produces blunt-ended PCR products) is used for amplification, a subsequent A-tailing step is mandatory. This involves an additional incubation of the purified blunt-ended PCR product with a non-proofreading polymerase (like Taq) and dATP.
2. Final Extension Step for A-Tailing: To ensure efficient and consistent 3'-A overhang generation, even when using Taq-based polymerases, a dedicated final extension step during the PCR cycle is highly recommended. An incubation at 72°C for 5-10 minutes (or a minimum of 5 minutes) at the end of the PCR program promotes complete A-tailing of the amplified fragments.
3. PCR Product Purification: It is strongly advised to purify the target PCR product prior to ligation. Gel purification using a commercially available kit, such as Bioneer's AccuPrep® PCR/Gel Purification Kit (Cat. No. K-3038), is the recommended method. Purification serves to remove contaminants such as unused primers, primer-dimers, dNTPs, polymerase, and salts from the PCR reaction mix, as well as to isolate the specific DNA band of interest from any non-specific amplification products. Using unpurified PCR products can significantly reduce cloning efficiency. This is likely because contaminants can compete for ligation with the vector, inhibit the T4 DNA Ligase, or contribute to a high background of undesirable clones. For instance, primer-dimers, if A-tailed, could ligate into the vector, leading to false positives or a high number of non-recombinant (blue) colonies if they are too small to effectively disrupt the lacZα gene.
4. Choice of Elution Buffer: When eluting the purified PCR product from a purification column or gel matrix, care should be taken regarding the composition of the elution buffer. The user manual for the AccuRapid™ TA Cloning Kit specifically cautions that commercially available elution buffers may contain EDTA, which can reduce ligation efficiency. T4 DNA Ligase requires Mg2+ as a crucial cofactor for its enzymatic activity. EDTA is a strong chelating agent that sequesters divalent cations like Mg2+. If EDTA is carried over from the purified PCR product into the ligation reaction, it can inhibit the ligase by depleting the available Mg2+, leading to failed or inefficient ligation. Therefore, using an elution buffer without EDTA (e.g., nuclease-free water or a low-salt Tris buffer) is preferable.
The ligation step
is where the A-tailed PCR insert is covalently joined to the T-tailed pBHA-T vector. The AccuRapid™ TA
Cloning Kit is optimized for a rapid ligation reaction.
1.
Reaction
Setup:
The user manual for the K-7170 kit provides details for setting up the ligation
reaction. A typical reaction mixture for a single sample would be assembled as
follows:
Table 3: Ligation Reaction
Setup for AccuRapid™ TA Cloning Kit (K-7170)
|
Component |
Recommended Amount/Volume (for a 10 µl total reaction) |
Key Considerations |
|
pBHA-T vector (25 ng/µl) |
1 µl (25 ng) |
Use the recommended amount.
Ensure vector integrity by proper storage and handling to preserve
T-overhangs. |
|
Purified PCR Product
(Insert) |
Variable (X µl) |
The optimal amount of insert depends on its size and concentration, as
achieving an appropriate molar ratio of insert to vector is crucial. While
general TA cloning protocols often suggest starting with an insert-to-vector
molar ratio ranging from 1:1 to 10:1, the K-7170 kit demonstrates optimal
cloning efficiency at 5:1 or 10:1 insert-to-vector molar ratios. For example,
25 ng of a ~2.7 kb vector (such as pBHA-T)
corresponds to approximately 0.013 pmol. To achieve
a 5:1 molar ratio (insert:vector),
approximately 0.065 pmol of insert is needed. This
equates to about 43–45 ng for a 1 kb insert. For a 10:1 ratio (insert:vector), this would be
approximately 0.13 pmol of insert, or about 85–90
ng for a 1 kb insert. |
|
AccuRapid™ 2X Reaction Buffer |
5 µl |
This buffer contains ATP and
Mg2+, essential for ligase activity. Avoid repeated freeze-thaw cycles of the
buffer. |
|
T4 DNA Ligase (200 U/µl) |
1 µl (200 Weiss units) |
This is a high concentration
of ligase, contributing to the rapid reaction time. Ensure enzyme is properly
stored at -20°C and handled on ice. |
|
Nuclease-free water |
To bring total volume to 10
µl |
Use high-quality, sterile,
nuclease-free water. |
Set up a positive control using 1 µl of the Control Insert instead of the PCR product. A negative control, without insert DNA, helps assess vector self-ligation levels.
2. Incubation Conditions: Once the reaction components are mixed gently (e.g., by flicking the tube and brief centrifugation), the ligation mixture is incubated at 25°C for 15 minutes. This combination of a relatively high incubation temperature (compared to traditional 16°C ligations) and a short incubation time is a key advantage of the "AccuRapid" system, significantly reducing the overall cloning workflow duration. After incubation, the ligation reaction mixture can be used immediately for transformation or stored on ice for short periods, or at -20°C for longer-term storage prior to transformation.
The
efficiency of the ligation reaction is highly sensitive to the molar ratio of
insert to vector. If too little insert is
used, the yield of recombinant plasmids will be low, potentially resulting in a
higher proportion of blue colonies (due to self-ligated or uncut vector). Conversely,
if too much insert is added, it can lead to the formation of concatemers
(multiple inserts ligated together) or may even inhibit the ligation reaction
itself. The K-7170 kit documentation recommends molar ratios of 5:1
or 10:1 (insert:vector) as
effective starting points for achieving high ligation efficiency. These ratios
have been shown to consistently yield a greater number of recombinant colonies.
Users should adjust the amount of insert based on the size and concentration of
their specific PCR product.
Following the
ligation reaction, the recombinant plasmids must be introduced into competent E. coli host cells for propagation and
selection.
1. Transformation: The entire 10 µl ligation reaction mixture is typically added to a tube of chemically competent E. coli cells. Standard heat-shock transformation protocols (e.g., incubation on ice, heat shock at 42°C, recovery in SOC or LB medium) are generally used. Alternatively, electroporation can be employed if electrocompetent cells and appropriate equipment are available. The choice of E. coli host strain is important; strains like DH5α, XL1-Blue, or TOP10 are commonly used as they are suitable for blue/white screening due to their lacZΔM15 genotype, which allows for α-complementation by the lacZα fragment encoded on the pBHA-T vector. The kit explicitly states that competent cells are an additional material required but not provided. The efficiency of transformation is a critical determinant of the number of colonies obtained. Low-quality competent cells or a suboptimal transformation procedure will result in few or no colonies, even if the ligation reaction was successful.
2. Plating and Selection: After a recovery period (typically 45-60 minutes at 37°C with shaking), the transformed cells are plated onto Luria-Bertani (LB) agar plates. These plates must contain ampicillin (e.g., 50-100 µg/ml) to select for cells that have successfully taken up the pBHA-T plasmid, which carries the ampicillin resistance gene.
3. Blue/White Screening: To differentiate between colonies harboring recombinant plasmids (with insert) and those with non-recombinant plasmids (empty vector), blue/white screening is employed. This requires the LB agar plates to be supplemented with X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) and IPTG (isopropyl-β-D-1-thiogalactopyranoside). IPTG induces the expression of the lacZα gene fragment from the plasmid. If no insert is present in the cloning site within lacZα, the α-fragment is produced, complements the ω-fragment produced by the host E. coli, and forms a functional β-galactosidase enzyme. This enzyme hydrolyzes X-gal, producing a blue-colored product, resulting in blue colonies. If a PCR product has been successfully ligated into the cloning site, it disrupts the lacZα coding sequence, preventing the production of a functional α-fragment. Consequently, no functional β-galactosidase is formed, X-gal is not hydrolyzed, and the colonies appear white. These white colonies are putative recombinants and are selected for further analysis.
The
clarity of blue/white screening can be influenced by factors such as the
freshness and concentration of X-gal and IPTG, the specific E. coli host strain used, the incubation
time and temperature of the plates, and the size of the insert. Very small
inserts might not fully disrupt the lacZα
reading frame or function, potentially leading to pale blue colonies that may
still contain the desired insert. Therefore, it is often advisable to analyze a
few light blue colonies in such cases, in addition to white colonies.
The Bioneer AccuRapid™ TA Cloning Kit (K-7170) is reported to
facilitate a high yield of recombinant clones. Experimental data presented by
the manufacturer indicates successful cloning across a range of insert sizes,
specifically tested with PCR products from 500 base pairs (bp) to 2,000 bp.
This comparative data suggests that the AccuRapid™ TA
cloning system performs favorably against at least one other supplier's TA
cloning kit in terms of the number of recombinant clones obtained within this
insert size range.
While the kit has
been validated for inserts up to 2,000 bp, the efficiency of TA cloning, in
general, tends to decrease as insert size increases further. This is often
attributed to steric hindrance affecting the ligation of larger DNA molecules
and the reduced molar concentration of larger inserts at a given mass
concentration. The provided documentation does not
specify an absolute maximum insert size limit for the K-7170 kit. Users
attempting to clone inserts significantly larger than 2 kb may need to optimize
ligation conditions further, such as increasing the insert concentration or
ligation time, and should anticipate potentially lower cloning efficiencies.
It is important
to note that the term "high number of recombinants" is a qualitative
descriptor. The available documentation does not provide quantitative metrics
for cloning efficiency, such as the percentage of white colonies that are
confirmed to contain the correct insert, or the number of colony-forming units
(CFU) per microgram of vector DNA. Such quantitative data would offer a more
rigorous basis for comparing performance and setting user expectations.
A primary
advantage of the AccuRapid™ TA Cloning Kit is the
significant reduction in the time required for the cloning process,
particularly at the ligation stage. The ligation reaction is completed in just
15 minutes at 25°C. This rapid protocol contrasts sharply with many traditional
DNA ligation methods that often necessitate incubations of several hours to
overnight, frequently at lower temperatures (e.g., 16°C or 4°C). This
acceleration of the ligation step contributes to a more streamlined and time-efficient
overall cloning workflow.
The convenience
of the kit is further enhanced by the core principle of TA cloning itself. PCR
products amplified with Taq polymerase (or similar A-tailing polymerases) can be used
directly for cloning after purification, without the need for restriction
enzyme digestion of the insert or the incorporation of restriction enzyme sites
into PCR primers. This simplifies the experimental
design and execution, especially when cloning fragments with unknown sequences
or lacking suitable restriction sites. This ease of use makes the AccuRapid™ TA Cloning Kit an attractive option for
researchers performing high-throughput cloning
projects or for those who may be less experienced with more complex, multi-step
cloning strategies that rely on restriction enzymes.
The AccuRapid™ TA Cloning Kit (K-7170) incorporates several
features within its pBHA-T vector that enhance its
utility for clone selection, screening, and downstream manipulation:
1. Blue/White Screening: The pBHA-T vector contains the lacZα gene, enabling visual differentiation of recombinant (white) colonies from non-recombinant (blue) colonies when plated on appropriate media containing IPTG and X-gal with a suitable E. coli host strain. This is a standard and widely used method for quickly identifying putative positive clones.
2. Convenient Excision of Inserts: The cloning site within the pBHA-T vector is flanked by BamHI restriction enzyme sites. This design allows for the straightforward excision of the cloned insert using a single BamHI digestion. This feature is particularly useful if the cloned fragment needs to be subcloned into a different vector system that has compatible BamHI sites.
3. Convenient Sequencing: The presence of M13 forward and M13 reverse primer binding sites, as well as T7 and SP6 promoter sequences, flanking the cloning site in the pBHA-T vector facilitates the sequencing of the cloned insert. Standard M13 primers can be used to obtain sequence information from both ends of the insert, allowing for verification of its identity, orientation, and the absence of PCR-induced mutations.
The
combination of ampicillin resistance for selection of transformants and
blue/white screening for identification of recombinants provides a robust
two-tiered system for isolating desired clones. However, it is crucial to
remember that blue/white screening is a presumptive test. White colonies should
always be further verified by independent methods, such as colony PCR,
restriction enzyme digestion of plasmid DNA, or DNA sequencing, to confirm the
presence and identity of the correct insert. This is because phenomena such as
mutations within the lacZα gene
itself or the ligation of very small, non-target DNA fragments can occasionally
lead to the formation of white colonies that do not contain the intended
recombinant construct.
For successful
cloning with the AccuRapid™ TA Cloning Kit (K-7170),
the insert DNA must meet specific criteria:
● Source: The insert must be a PCR product.
● 3'-End Structure: The PCR product must possess single 3'-deoxyadenosine (A) overhangs. These are typically generated when using Taq DNA polymerase or other non-proofreading DNA polymerases that exhibit terminal transferase activity. A dedicated final extension step (e.g., 72°C for 5-10 minutes) during PCR is recommended to ensure efficient A-tailing.
● Purification: Purification of the PCR product, preferably by gel extraction, is highly recommended to remove primers, dNTPs, polymerase, and non-specific products, as these can significantly inhibit cloning efficiency. Elution buffers containing EDTA should be avoided.
● Size: The kit has been demonstrated to be effective for cloning inserts ranging from 500 bp to 2,000 bp. Performance with inserts outside this range, particularly much larger ones, may require optimization.
● "Freshness": While not explicitly detailed for K-7170, general best practices for TA cloning suggest that freshly prepared PCR products yield higher efficiencies. The 3'-A overhangs can degrade over time or with repeated freeze-thaw cycles, potentially reducing the number of suitable molecules for ligation.
The pBHA-T vector provided in the kit has the following key
specifications:
● Type: Linearized plasmid vector specifically prepared for TA cloning.
● 3'-End Structure: Possesses single 3'-deoxythymidine (T) overhangs, complementary to the 3'-A overhangs of suitable PCR products.
● Approximate Size: While not explicitly stated, pUC-based TA cloning vectors are typically around 2.7 kb (e.g., pMD18-T). The features listed for pBHA-T are consistent with this class of vectors.
● Key Features: Contains the lacZα gene for blue/white screening, an ampicillin resistance gene for selection, M13 forward/reverse primer sites and T7/SP6 promoter sites for sequencing and/or in vitro transcription, BamHI sites flanking the insert for easy excision, and a pUC origin of replication for high-copy number amplification in E. coli.
● Product Code: K-7170.
● Available Size: The standard kit size is 20 reactions.
● Components: As detailed in Table 1 (Section II.B), the kit includes pBHA-T vector, Control Insert, AccuRapid™ 2X Reaction Buffer, and T4 DNA Ligase.
● Shipping Condition: The product is shipped on dry ice to maintain the stability of its enzymatic and DNA components. This underscores the temperature sensitivity of the reagents, particularly the T4 DNA Ligase and the pre-linearized T-vector, and necessitates immediate transfer to appropriate storage upon receipt.
Proper storage is
essential to maintain the performance and shelf life of the AccuRapid™
TA Cloning Kit:
● Storage Temperature: All components of the kit should be stored at -20°C upon arrival and when not in use.
● Freeze-Thaw Cycles: Repeated freeze-thaw cycles should be avoided, especially for the pBHA-T vector and the AccuRapid™ 2X Reaction Buffer. To minimize potential degradation and loss of efficiency, it is recommended to aliquot these components into smaller, single-use volumes if the entire amount will not be consumed in a short period. For the pBHA-T vector, freeze-thaw cycles can physically damage the delicate single-stranded T-overhangs or expose them to nuclease activity. For the 2X Reaction Buffer, which contains ATP, repeated freeze-thaw cycles can lead to ATP hydrolysis, reducing the energy source available for the T4 DNA Ligase and thereby diminishing ligation efficiency.
● Shelf Life: The provided documentation does not explicitly state a specific shelf life or expiration date for the K-7170 kit. Bioneer states that it "guarantees quality during the warranty period as specified", but the duration of this warranty period is not defined in the available materials. Users should typically refer to the product packaging for an expiry date or contact the manufacturer for this information if it is not readily available.
Even with
optimized protocols, issues can arise during molecular cloning. The following
table outlines common problems encountered when using TA cloning kits like the AccuRapid™ TA Cloning Kit (K-7170), along with their
potential causes and suggested solutions. This information is synthesized from
the Bioneer manual for K-7170 1 and supplemented with general
cloning troubleshooting advice.
Table 4: Troubleshooting Guide for the AccuRapid™
TA Cloning Kit
|
Observed Problem |
Potential Cause(s) |
Suggested Solution(s) |
|
1. No colonies (or very few colonies) produced |
Competent Cells/Transformation: Low transformation efficiency of competent cells.
Competent cells mishandled (e.g., thawed improperly, incorrect heat shock).
Incorrect antibiotic on plates or antibiotic
degraded. Too little DNA transformed. Ligation
Failure: Inactive T4 DNA Ligase (e.g., improper storage, expired).
Degraded ATP in 2X Reaction Buffer (e.g., multiple freeze-thaws). Inhibitors
present in ligation reaction (e.g., EDTA from PCR purification, high salt).
Problems with vector (degraded T-overhangs) or insert (no A-overhangs,
degraded). Incorrect ligation temperature or time (though less likely with
this kit's robust protocol). |
Competent Cells/Transformation: Test competent cell efficiency with a control plasmid
(e.g., pUC18). Prepare or purchase fresh, high-efficiency cells. Strictly
follow the recommended transformation protocol. Use fresh agar plates with
the correct concentration of ampicillin (e.g., 50-100 µg/ml).
Ensure sufficient amount of ligation product is
added to cells. Ligation Failure:
Use fresh ligase or new kit components. Ensure proper -20°C storage. Aliquot
2X Reaction Buffer to avoid multiple freeze-thaws. Ensure PCR product is thoroughly purified; avoid EDTA in final
elution. Re-prepare fresh PCR insert with proper A-tailing. Use fresh vector.
Run positive control ligation (with kit's Control Insert). |
|
2. Most colonies are blue (or light blue); few or no
white colonies |
Insert Issues:
PCR product lacks 3'-A overhangs (e.g., used proofreading polymerase without
A-tailing step). Insufficient amount of PCR insert
in ligation (unfavorable insert:vector
molar ratio). PCR insert degraded or A-overhangs lost (e.g., old PCR product,
nuclease contamination). PCR product purification method removed A-overhangs.
Vector Issues: High background of
non-recombinant vector (e.g., self-ligation of vector if T-overhangs are
damaged or if some vector molecules were not properly linearized/T-tailed
during manufacturing). Screening
Issues: Insert is very small (<500 bp) and may not fully disrupt lacZα function, leading to pale blue
colonies that could still be recombinant. |
Insert Issues:
Ensure use of Taq or other
A-tailing polymerase with adequate final extension. If using proofreading
polymerase, perform a separate A-tailing step. Optimize insert:vector molar ratio; try increasing the
amount of insert. Use freshly prepared and purified PCR product.
Verify that the purification method (especially gel extraction) does not
damage or remove overhangs. Vector
Issues: This is less controllable by the user for a pre-prepared T-vector, but ensure proper storage of the vector. A
negative control (vector + ligase, no insert) can assess self-ligation
levels. Screening Issues: If expecting small inserts, pick and analyze some light
blue colonies in addition to white ones. |
|
3. White colonies do not contain insert
DNA (or contain incorrect/multiple inserts) |
Vector Issues:
Vector degradation (loss of T-overhangs) leading to blunt-end ligation of
vector to itself, potentially with small deletions/mutations in lacZα causing a white phenotype.
Spontaneous mutations in the lacZα
gene of the vector. Insert Issues:
Ligation of primer-dimers or small, non-specific PCR products that were not
fully removed during purification, if they disrupt lacZα. Ligation of multiple insert fragments (concatemers) if
insert concentration was too high. Contamination:
Contamination of PCR product or ligation reaction with other DNA fragments. |
Vector/Insert/Contamination Issues: Always perform careful gel purification of the PCR
product to isolate only the band of the correct size. Optimize insert:vector molar ratio; avoid
excessive insert concentrations. Screen multiple white colonies by colony PCR
using insert-specific primers, or by restriction digestion of miniprep
plasmid DNA. Sequence putative positive clones to confirm insert identity and
integrity. Maintain good laboratory practice to prevent DNA contamination. |
Many
troubleshooting scenarios ultimately point back to the critical importance of
the input materials: a high-quality, correctly A-tailed, and thoroughly
purified PCR insert, and an intact pBHA-T vector with
functional T-overhangs. Meticulous attention to the preparation and handling of
these components, along with careful execution of the ligation and
transformation steps, will significantly increase the probability of successful
cloning with the AccuRapid™ TA Cloning Kit.
The Bioneer AccuRapid™ TA Cloning Kit (K-7170) is a system designed for
the straightforward and rapid cloning of PCR products that possess 3'-A
overhangs. Its core mechanism relies on the efficient ligation of these inserts
into the provided pBHA-T vector, which is
pre-linearized and features complementary 3'-T overhangs, catalyzed by T4 DNA
Ligase.
Key
distinguishing features of this kit include:
● Speed: A remarkably fast ligation reaction time of 15 minutes at 25°C.
● Convenience: Simplifies cloning by eliminating the need for restriction enzyme digestion of PCR products or the incorporation of restriction sites into primers.
● Vector Features: The pBHA-T vector incorporates essential elements for successful cloning, including the lacZα gene for blue/white screening, an ampicillin resistance marker for selection, M13 primer sites for sequencing, and flanking BamHI sites for easy insert excision.
● Performance: The kit is reported to be effective for cloning inserts in the 500 bp to 2,000 bp range, with comparative data suggesting good efficiency.
The
AccuRapid™ TA Cloning Kit (K-7170) appears to be a
well-designed implementation of standard TA cloning methodology, with its
primary competitive differentiation being the significantly reduced ligation
time. This makes it a valuable tool for researchers seeking to accelerate their
cloning workflows without resorting to more complex or expensive cloning
technologies.
To maximize the
success rate and reliability when using the Bioneer AccuRapid™
TA Cloning Kit (K-7170), the following considerations are paramount:
1. PCR Product Quality:
○ Ensure the use of a DNA polymerase that generates 3'-A overhangs (e.g., Taq polymerase). Include an adequate final extension step (5-10 minutes at 72°C) in the PCR protocol to maximize A-tailing efficiency.
○ Thoroughly purify the PCR product, preferably by gel extraction, to remove contaminants that can inhibit ligation or lead to unwanted side products. Avoid elution buffers containing EDTA, as this can chelate Mg2+ and inhibit T4 DNA Ligase.
2. Ligation Reaction Optimization:
○ While the kit provides
a streamlined protocol, the molar ratio of insert to vector is a critical
parameter. For experimental inserts, empirical optimization may still be
necessary. According to the K-7170 kit documentation, starting with an
insert-to-vector molar ratio of 5:1 or 10:1 is recommended to achieve optimal
ligation efficiency and maximize the number of recombinants.
3. Transformation Efficiency:
○ Use high-quality competent E. coli cells with a known high transformation efficiency. Adhere strictly to the recommended transformation protocol for the chosen cells.
4. Kit Component Integrity:
○ Store all kit components at -20°C immediately upon receipt and when not in use.
○ To preserve the activity of the T4 DNA Ligase and the integrity of the pBHA-T vector's T-overhangs and the ATP in the 2X Reaction Buffer, avoid repeated freeze-thaw cycles by aliquoting these components into single-use volumes.
5. Verification of Clones:
○ Blue/white screening is a presumptive test. Always verify putative white (recombinant) colonies by independent molecular methods, such as colony PCR using insert-specific or vector-based primers, restriction enzyme digestion of isolated plasmid DNA, and ultimately, DNA sequencing of the insert.
While
the AccuRapid™ TA Cloning Kit (K-7170) offers
significant convenience and speed for the ligation step, the overall success of
the cloning experiment remains heavily dependent on the user's proficiency in
fundamental molecular biology techniques. Careful PCR optimization, meticulous
DNA purification, robust bacterial transformation, and diligent clone analysis
are indispensable for achieving desired outcomes. The kit provides effective
tools for a critical part of the cloning workflow, but it cannot compensate for
deficiencies in upstream preparation or downstream handling and analysis.