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In‑Fusion Cloning FAQs

The following FAQs apply to current In‑Fusion Cloning kits: In‑Fusion HD Cloning Plus, In‑Fusion HD Cloning Plus CE, and In‑Fusion HD EcoDry Cloning Plus.

General Information

What is In‑Fusion Cloning?

In‑Fusion Cloning is a highly efficient, ligation-independent cloning method based on the annealing of complementary ends of a cloning insert and linearized cloning vector. This technology ensures easy, single-step directional cloning of any gene of interest into any vector at any locus. In‑Fusion constructs are seamless, enabling translational reading frame continuity without any interfering “scar” sequences.

What is the efficiency of In‑Fusion Cloning?

Cloning efficiency is at least 95% for a single insert into a vector. Unlike transformation efficiency, which is merely a measure of the number of transformed colonies obtained, cloning efficiency is a measure of accuracy, providing information on the number of correct clones obtained from a cloning reaction.

How does In‑Fusion Cloning work?

In‑Fusion Cloning requires 15 bp of overlap at the termini of the cloning insert and linearized cloning vector, or adjacent cloning inserts if multiple inserts are to be joined simultaneously.

These 15-bp homologous overlaps can be generated by PCR amplification or oligo synthesis of either of the cloning components. Homologous overlaps shorter than 12 nt or longer than 21 nt are not recommended. Translational reading frame continuity of a fusion construct can be adjusted by adding nucleotides between the insert-specific sequence and 15-nt overlap. 15-bp complementary regions must be located at the termini of adjacent DNA fragments or they will not be joined by In‑Fusion Cloning.

The In‑Fusion enzyme mix generates 15-nt single-stranded 5' overhangs at the termini of the cloning insert and linearized cloning vector. These overhangs are annealed at the sites of complementarity, and the recombinant circular construct is rescued in E. coli. (We do not recommend use of cells with competency less than 108 cfu/µg supercoiled DNA.) In‑Fusion Cloning does not allow for the covalent assembly of linear DNA molecules.

The In-Fusion protocol clones any insert into any vector in as little as 15 min

A brief overview of the In‑Fusion Cloning protocol.

What is the difference between In‑Fusion HD Cloning Plus and In‑Fusion HD EcoDry Cloning Plus?

In‑Fusion HD Cloning Plus includes liquid In‑Fusion HD Enzyme Premix, whereas In‑Fusion HD EcoDry Cloning Plus includes pre-aliquoted, lyophilized In‑Fusion HD Enzyme Premix. The EcoDry kit minimizes handling and stores at room temperature. The liquid cloning reaction is complete in 15 min, and the EcoDry cloning reaction is complete in 30 min.

What is Cloning Enhancer (CE)?

Cloning Enhancer, or CE, is a proprietary enzyme mix for removing background plasmid DNA and PCR residue, thus eliminating the need for additional purification of PCR-amplified DNA prior to the In‑Fusion reaction. (See details in the In‑Fusion HD Cloning Kit User Manual.) CE is available as part of the In‑Fusion HD Cloning Plus CE kit and as a separate item.

Use of CE is only appropriate if PCR amplification generates a single PCR fragment of the expected size, without a background smear. CE is a convenient tool for high-throughput applications that employ highly optimized PCR cycling conditions and primers that generate clean DNA fragments of the expected size.

The addition of CE to the In‑Fusion reaction mix is not required, nor does it increase cloning efficiency—it simply replaces standard purification steps, provided that PCR gives high-quality results.

Does the In‑Fusion Cloning method introduce errors into the sequence?

Clontech has not seen any base slippage, base addition, or base deletion with the In‑Fusion Cloning enzyme. We have cloned and sequenced over 4,000 separate clones and various human open reading frames subsequent to In‑Fusion Cloning, and have rarely seen any evidence of errors at the cloning junctions (<2%). Most of the sequence errors that we have come across are clearly due to errors in primer synthesis (that is, they appear in all or many of the clones containing a particular insert). In‑Fusion Cloning is ideal for making error-free fusion constructs.

How stable is the In‑Fusion Cloning enzyme mix?

The In‑Fusion HD cloning enzyme mix has been engineered for increased stability, requires no dilution, and can be stored at –20°C (liquid) or room temperature (EcoDry).

Inserts and Primer Design

What are the requirements for a homologous overlap that will facilitate a successful In‑Fusion Cloning reaction?

Homologous overlaps are necessary for In‑Fusion Cloning. Appropriate homology consists of a 15-nt DNA sequence complementary to the 5' end of a linearized cloning vector or cloning insert. Figures 2 and 3 in the In‑Fusion HD Cloning Kit User Manual or In‑Fusion HD EcoDry Cloning Kit User Manual provide detailed examples.

How do I generate homologous overlaps between the termini of cloning inserts and linearized vectors?

Homologous inserts are created through PCR amplification of cloning inserts using primers specifically designed to incorporate 15 nt of 5' overhangs complementary to the termini of the linearized vector (or adjacent cloning insert).

Alternatively, 15 nt of homology may be added to a vector linearized via inverse PCR, such that it overlaps with the cloning insert. If synthetic oligonucleotides (≥50 bp) are being cloned, these oligos may carry the 15-nt 5' overhangs homologous to the ends of the linearized cloning vector or adjacent DNA fragments. (High-quality, non-phosphorylated oligos are compatible with In‑Fusion Cloning.)

How do I design PCR primers carrying 15-nt overhangs complementary to the termini of the linearized vector or adjacent insert?

Each forward (5' → 3' sense strand) and reverse (5' → 3' antisense strand) PCR primer should include the following:

  • A template-specific (gene-specific) portion at its 3' end. To ensure specific and efficient PCR amplification, the template-specific portion of the primer should be 18–25 nt in length.
  • 15 nt of homology at the 5' end of the primer, complementary to the termini of the linearized vector or adjacent inserts (if multiple inserts are to be cloned simultaneously). Homologous overlaps shorter than 12 nt and longer than 21 nt are not recommended. The 15-bp complementary regions must be located at the termini of adjacent DNA fragments or they will not be joined by In‑Fusion Cloning.
  • (Optional) To ensure continuity of the translational reading frame, or to preserve restriction site(s), additional nucleotides can be added to the PCR primer(s) between the template-specific portion and the 15-nt homologous overlap.

What is the optimal length of the homologous overlap between the termini of the PCR-amplified insert and linearized cloning vector?

Current In‑Fusion reaction conditions favor 15 bp of homologous overlap. We do not recommend using overlaps shorter than 12 bp or longer than 21 bp.

What tools are available to assist in the design of PCR primers compatible with In‑Fusion Cloning?

Instructions for designing In‑Fusion PCR primers are included in all In‑Fusion Cloning user manuals. Additionally, our online Primer Design Tool facilitates primer design for single- and multiple-fragment cloning, and is compatible with Mozilla Firefox or Google Chrome web browsers. (Internet Explorer is not compatible with the Primer Design Tool.) Step-by-step tutorials for the Primer Design Tool are also available in the Cloning Resources section of our website.

We also recommend SnapGene Viewer as a helpful, free online tool for in silico assembly of your recombinant construct, manual design of In‑Fusion PCR primers, and adjustment of translational reading frame continuity.

Why are homologous overlaps important for In‑Fusion Cloning reactions?

The mechanism for In‑Fusion Cloning reactions employs a 3' exonuclease to generate single-stranded 5' overhangs at the termini of linear double-stranded DNA. These DNA fragments are then annealed via complementary 15-nt overlaps at the termini of the insert(s) and a linearized vector. The vector can be linearized by inverse PCR or restriction digest. Restriction digest can be performed with one or more enzymes that generate 5' overhangs (e.g., EcoRI, BamHI), 3' overhangs (e.g., KpnI), or blunt ends (e.g., HpaII).

The diagrams below show specific examples of the In‑Fusion Cloning mechanism in action:

Mechanism of the In-Fusion Cloning reaction for a linearized vector with 5’ and 3’ overhangs Mechanism of the In-Fusion Cloning reaction for a linearized vector with a 5’ overhang and blunt end

How do I calculate the 15-nt overlap if the vector is linearized via restriction digest, generating a 5' or 3' overhang?

The 5' overhang of a restriction site is included in the 15-nt complementary region. The 3' overhang of a restriction site is excluded from the 15-nt complementary region. Figures 2 and 3 in the In‑Fusion HD Cloning Kit User Manual or In‑Fusion HD EcoDry Cloning Kit User Manual provide detailed examples.

Restriction sites used for vector linearization can be preserved in the recombinant vector by adding nucleotides to the PCR primers between the template-specific portion and the 15-nt homologous overlap. The online Primer Design Tool allows you to choose whether or not to preserve the restriction sites. (The Primer Design Tool is compatible with Mozilla Firefox or Google Chrome web browsers, but not with Internet Explorer.)

How can I alter the reading frame when performing In‑Fusion Cloning?

The reading frame is defined by the primer sequence. For example, when creating a fusion protein, if the 15 bp of vector homology at the 5' end of the In‑Fusion PCR primer sequence corresponds to the last five codons of the vector reading frame, you would clone your new gene or tag in the same reading frame downstream of the C-terminus of the vector sequence by placing the first codon of this gene next to the last codon of the homology sequence (i.e., at the 3' end) without any interfering STOP codons. To shift the reading frame, you would simply add one or two additional bases after the 15-bp homology and before the first codon of the target gene. For example:

5' 15-nt homology with vector sequence Number of bases needed to maintain reading frame 3' gene-specific sequence of the In‑Fusion PCR primer
GTA TTC ATC CGG CCG 0 ATG GGC CTT TAC CCA ACT CGC
G TAT TCA TCC GGC CG 1 ATG GGC CTT TAC CCA ACT CGC
GT ATT CAT CCG GCC G 2 ATG GGC CTT TAC CCA ACT CGC

How do I clone my gene of interest in the same translational reading frame as a tag present in the cloning vector (e.g., fluorescent protein, Myc, HA, etc.)?

Translational reading frame continuity with a tag is adjusted within the length of the gene-specific portion of the PCR primer, or by adding nucleotides between the gene-specific portion and the 15-nt homology of the PCR primer.

Please note that the current version of our online Primer Design Tool does not allow an adjustment for translational reading frame continuity. As such, the primer sequence should be manually designed by the user; we recommend SnapGene Viewer as a helpful, free online tool to help with this task.

Can small external sequences be included in the In‑Fusion PCR primer?

Yes—external nucleotide sequences (e.g., small tags, Kozak sequences, restriction sites, etc.) can be added between the template/gene-specific portion and the 15-nt homologous overlap of the In‑Fusion PCR primer.

Our In‑Fusion Webinar Series includes a pre-recorded video specific to this application.

For In‑Fusion Cloning, is it a problem if the 15-bp region of homology is present more than once in the vector? Will multiple recombination products result?

Internal recombination events at sites other than those adjacent to the vector linearization site are extremely rare. Therefore, even if your desired region of homology is present more than once in the vector sequence, unwanted recombination events are unlikely to occur.

Do I need to use phosphorylated PCR primers for In‑Fusion Cloning?

No—the use of phosphorylated oligonucleotides is not required for In‑Fusion Cloning.

What oligonucleotide quality is required for an In‑Fusion PCR primer?

In‑Fusion PCR primers should be high-quality oligonucleotides, purified by desalting. Gel or HPLC purification is not required.

What PCR polymerases are recommended for amplification of the In‑Fusion cloning insert?

In‑Fusion Cloning is compatible with any PCR polymerase. 3' overhangs do not interfere with the cloning reaction.

To ensure an error-free insert, use a polymerase with high proofreading activity, like CloneAmp HiFi PCR Premix (supplied with all current In‑Fusion Cloning kits). This polymerase is highly robust and accurate, enabling amplification of up to 6 kb of human genomic DNA, 10 kb of E. coli genomic DNA, and 15 kb of Lambda DNA. It is compatible with two- or three-step PCR cycling, and exhibits minimal error rates on GC-rich templates.

CloneAmp HiFi Polymerase preserves the integrity of your cloning insert and vector

Mutation frequency of CloneAmp HiFi Polymerase compared to other high-fidelity PCR enzymes. Eight arbitrarily selected GC-rich regions were amplified with CloneAmp HiFi Polymerase or other DNA polymerases using a Thermus thermophilus HB8 genomic DNA template, and cloned into suitable plasmids. Multiple clones were selected for each amplification product and subjected to sequence analysis. DNA fragments amplified using CloneAmp HiFi Polymerase yielded only 12 mismatched bases per 542,580 total bases—lower than an alternative high-fidelity enzyme from Company A, and 10-fold lower than Taq DNA polymerase.

Do PCR-generated 3' A-overhangs interfere with In‑Fusion Cloning?

No, 3' A-overhangs do not interfere with the In‑Fusion Cloning mechanism.

Can I clone multiple fragments into one vector in a single In‑Fusion Cloning reaction?

Yes—we have successfully tested multiple-fragment cloning with up to five inserts. (See Figure and Table below for cloning schematic and colony screen results, respectively).

Primer design for multiple-fragment cloning can be done with our online Primer Design Tool. (The Primer Design Tool is compatible with Mozilla Firefox or Google Chrome web browsers, but not with Internet Explorer.) We also have a Primer Design Tool tutorial specifically for multiple-fragment cloning.

Please note that between two adjacent fragments, only one homologous overlap is required for the In‑Fusion reaction. This overlap can be located on either of the fragments.

Homologous overlaps facilitate seamless multiple-fragment cloning in a single reaction

The In‑Fusion HD Cloning System has an improved capability for cloning multiple fragments in a single reaction. Using this system, cloning up to four 1-kb fragments simultaneously is as easy as cloning a single fragment. This saves weeks that would otherwise be spent screening clones and subcloning.

Insert Colony Screening
Fragments Colonies, 1/5 plated Correct clones
1 kb + 1 kb 2,128 10/10
1 kb + 1 kb + 1 kb 83 7/10
1 kb + 1 kb + 1 kb + 1 kb 31 8/10
1 kb + 1 kb + 1 kb + 1 kb + 1 kb 14 4/10

Can I split the homologous 15-nt overlap between the insert and vector, or adjacent inserts?

Yes. The homologous 15-nt overlap can be split between adjacent DNA fragments. However, splitting the overlap between an insert and vector can only be done if the vector is linearized via inverse PCR.

Primer design for this option is not facilitated by the online Primer Design Tool. We recommend SnapGene Viewer as a helpful, free online tool to help with this task.

The diagram below shows In‑Fusion primer design and the annealing of complementary strands, using a 15-nt overlap split between Fragment 1 (red) and Fragment 2 (blue):

Schematic for splitting the homologous overlap between an insert and PCR-linearized vector

Do I have to purify the PCR-amplified insert and/or vector prior to performing the In‑Fusion Cloning reaction?

Yes, the PCR-amplified DNA must be purified prior to In‑Fusion Cloning. 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 (NucleoSpin Gel and PCR Clean‑Up), or treat your PCR product with Cloning Enhancer (CE). However, if non-specific background or multiple bands are visible on your gel, isolate your target fragment by gel extraction. If you use PCR to amplify your vector and insert and you obtain both a PCR-amplified vector and PCR-amplified fragment(s) without non-specific background, you can use the Quick In‑Fusion Cloning Protocol provided in Appendix A of the user manual.

  • NucleoSpin Gel and PCR Clean‑Up
    • Gel extraction enables selection of specific DNA fragments of the desired size from background PCR byproducts or other contaminants.
    • Column purification is appropriate if PCR did not produce a background smear.
  • Cloning Enhancer (CE)
    • This proprietary enzyme mix removes background plasmid DNA and PCR residue.
    • CE is appropriate for PCR that results in a single fragment of the expected size, without a background smear.
    • CE is a convenient tool for HTP applications that employ highly optimized PCR cycling conditions and primers such that PCR generates clean DNA fragments of the expected size.

Note: In most cases, CE treatment does not require additional column purification or gel extraction. However, to ensure better cloning results, PCR-linearized vectors may require a combination of CE treatment followed by gel extraction to separate a linearized vector from possible PCR byproducts.

What is the largest DNA fragment compatible with In‑Fusion Cloning?

This technology has been optimized for cloning large fragments. DNA inserts up to 15 kb have been successfully cloned into pUC19 using In‑Fusion Cloning.

High success rate for In-Fusion Cloning with large DNA fragments

Ten out of ten colonies contain the correct insert (100% efficiency) when cloning fragments as large as 15 kb (results confirmed by colony PCR screening).

What is the smallest DNA fragment compatible with In‑Fusion Cloning?

The smallest insert successfully cloned with In‑Fusion Cloning was a 50-bp synthetic oligonucleotide (including two 15-nt homologous overlaps with the vector termini).

For In‑Fusion Cloning of short synthetic oligos (between 50 and 150 bp), the suggested oligo to vector molar ratio is 5–15:1. Depending on oligo length, the optimal ratio must be determined empirically.

Note: Non-phosphorylated oligonucleotides are compatible with In‑Fusion Cloning. However, 3' exonuclease activity in the In‑Fusion enzyme mix requires terminal 3' OH groups.

Vectors

What cloning vectors are compatible with In‑Fusion Cloning?

Any linear vector is compatible with In‑Fusion Cloning. Linearization can be accomplished in one of the following ways:

  • Restriction digest with one or more restriction enzymes.
    • For efficient In‑Fusion Cloning, integrity of the linearized vector termini is essential. We recommend using high-quality restriction enzymes, and performing digests over several hours. However, overnight restriction digest is not advisable.
    • Dephosphorylation of the vector termini is not required; the vector will not re-circularize in the In‑Fusion Cloning reaction mix unless it carries 15-nt complementary overlaps at its termini.
  • Inverse PCR with primers positioned at the desired cloning site.
    • Choice of cloning locus is flexible since suitable restriction sites are not required.
    • Simultaneous PCR-mediated mutagenesis (deletion, insertion, base change) is possible. (Please see our prerecorded webinar on this application.)
    • The 15-bp homologous overlaps can be added to the PCR-linearized vector.
    • Preserve the integrity of the vector backbone by using a PCR polymerase with high proofreading activity, like CloneAmp HiFi PCR Premix (supplied with In‑Fusion HD Cloning Plus kits). This polymerase is highly robust and accurate, enabling amplification of up to 6 kb of human genomic DNA, 10 kb of E. coli genomic DNA, or 15 kb of Lambda DNA. It is compatible with two- or three-step PCR cycling, and exhibits minimal error rates on GC-rich templates. CloneAmp HiFi Polymerase preserves the integrity of your cloning insert and vector

      Mutation frequency of CloneAmp HiFi Polymerase compared to other high-fidelity PCR enzymes. Eight arbitrarily selected GC-rich regions were amplified with CloneAmp HiFi Polymerase or other DNA polymerases using a Thermus thermophilus HB8 genomic DNA template, and cloned into suitable plasmids. Multiple clones were selected for each amplification product and subjected to sequence analysis. DNA fragments amplified using CloneAmp HiFi Polymerase yielded only 12 mismatched bases per 542,580 total bases—lower than an alternative high-fidelity enzyme from Company A, and 10-fold lower than Taq DNA polymerase.

Vectors linearized via restriction digest should be purified by a preparative agarose gel (covered with aluminum foil to prevent DNA damage). Electrophoresis should be done at a low voltage to ensure the separation of linear and circular (uncut) vector molecules.

Vectors linearized via inverse PCR should be treated with Cloning Enhancer (CE) to destroy the parental plasmid. CE-treated, PCR-linearized vectors may require additional purification by agarose gel electrophoresis if PCR byproducts are present in the linearized vector prep.

Does In‑Fusion Cloning preserve the restriction site(s) used to linearize the vector?

In order to maintain the restriction sites, nucleotides can be added to the PCR primers between the template-specific portion and the 15-nt homologous overlap.

The online Primer Design Tool allows you to choose whether or not to preserve the restriction sites. (The Primer Design Tool is compatible with Mozilla Firefox or Google Chrome web browsers, but not with Internet Explorer.)

Do I have to dephosphorylate the termini of a linearized vector for In‑Fusion Cloning?

No, dephosphorylation of the vector termini is neither required nor recommended for In‑Fusion Cloning.

Are large cloning vectors compatible with In‑Fusion Cloning?

Yes, In‑Fusion technology allows easy cloning of single or multiple DNA fragments directly into large vectors (e.g., adenoviral vectors at 32.6–36 kb) in a single reaction, without intermediate cloning into transfer/shuttle vectors. (Please see Figures 1, 2, 5, and Table III of the Adeno-X Adenoviral System 3 Brochure for details.)

Is In‑Fusion Cloning compatible with vectors carrying repeated sequences?

Yes—Clontech scientists routinely use In‑Fusion Cloning to clone transgenes into lentiviral or retroviral vectors that carry long terminal repeats (LTRs), as well as adenoviral vectors that carry inverted terminal repeats (ITRs).

Applications

Can I use In‑Fusion Cloning to assemble a covalently linked linear DNA molecule?

No, In‑Fusion Cloning does not allow the assembly of covalently linked linear DNA molecules.

In‑Fusion Cloning kit components include a linearized cloning vector, enabling the rescue of a circular recombinant construct in E. coli.

Can I use a circular cloning vector for In‑Fusion Cloning?

No, circular cloning vectors are not compatible with In‑Fusion Cloning. A vector must be linearized via restriction digest or inverse PCR.

Can I use In‑Fusion Cloning to clone a DNA fragment generated by restriction digest?

Yes—if the adjacent DNA fragments/oligos or linearized vector carry the 15-bp homologous overlaps required for annealing. 15-bp overlaps with the digested cloning insert may be added to the termini of a PCR-linearized vector or a synthetic oligonucleotide.

Will In‑Fusion technology allow cloning of an insert if the sites of complementarity are located at a distance from the linearized vector termini?

No—homologous 15-bp overlaps should be located precisely at the termini of the vector and insert. 15-bp complementary regions not located at the termini of adjacent DNA fragments will not be joined by In‑Fusion Cloning. PCR linearization of a vector allows positioning of the primers at the desired cloning site, thus enabling the generation of the 15-bp overlaps at the termini.

Is In‑Fusion Cloning compatible with vectors carrying repeated sequences?

Yes—Clontech scientists routinely use In‑Fusion Cloning to clone transgenes into lentiviral or retroviral vectors that carry long terminal repeats (LTRs), as well as adenoviral vectors that carry inverted terminal repeats (ITRs).

Can I use In‑Fusion Cloning for mutagenesis?

Yes, In‑Fusion Cloning allows single or multiple base changes, deletions, and insertions. For details, please see our prerecorded mutagenesis webinar and/or the Mutagenesis with In‑Fusion HD Cloning Plus tech note.

Can I clone an oligonucleotide/shRNA oligonucleotide using In‑Fusion Cloning?

Yes, synthetic oligonucleotides (≥50 bp), carrying homologous overlaps with the termini of the linear vector, can be cloned using In‑Fusion Cloning. For cloning of short synthetic oligos (between 50 bp and 150 bp), the suggested oligo to vector molar ratio is 5–15:1. Depending on the oligo length, the optimal molar ratio must be determined empirically.

Note: Non-phosphorylated oligonucleotides are compatible with In‑Fusion Cloning. However, 3' exonuclease activity in the In‑Fusion enzyme mix requires terminal 3' OH groups.

Can I use In‑Fusion Cloning to clone GC-rich DNA fragments?

Yes, but special consideration should be given to the homologous overlaps of adjacent DNA fragments. Since In‑Fusion Cloning is based on the annealing of these overlaps, it is important to take the GC content into account for the 15-bp homology. We have no specific data showing variability of current In‑Fusion HD Cloning kit performance depending on the GC content of the 15-bp overlap. However, the following results were obtained using a previous version of the kit—In‑Fusion Advantage:

  • 15-bp homologous overlaps with GC content of 20–40% had little or no effect on the In‑Fusion Advantage cloning efficiency.
  • 15-bp homologous overlaps with GC content of 60–80% showed a reduced In‑Fusion Advantage cloning efficiency in certain cases.

Tips

For a more extensive discussion of In-Fusion Cloning Tips, please visit this page.

What are the recommended insert to vector molar ratios for In‑Fusion Cloning?

In‑Fusion HD Cloning Plus uses a very robust enzyme, and allows highly efficient cloning in most situations. General recommendations on insert/vector quantities are included in all current In‑Fusion Cloning user manuals.

  • To ensure optimal results under standard conditions, or when performing single- or multiple-fragment cloning, use an insert to vector ratio of 2:1.
    • The molar ratio of each of the multiple inserts should be 2:1 with regards to the linearized, purified vector. The molar ratio of two inserts with one vector should be 2:2:1.
    • To calculate the required amount of each of the DNA fragments, use no less than 20 ng of the smallest insert and calculate the quantities of the rest of the fragments accordingly, maintaining the 2:1 insert to vector molar ratio. (Each of the inserts should be calculated at the 2:1 molar ratio with regard to the vector.)
  • For cloning of small DNA fragments (between 150 and 350 bp), the suggested insert to vector molar ratio is 3–5:1.
  • For cloning of short synthetic oligos (between 50 bp and 150 bp), the suggested oligo to vector molar ratio is 5–15:1. Depending on the oligo length, the optimal molar ratio must be determined empirically.
    • Non-phosphorylated oligonucleotides are compatible with In‑Fusion Cloning. However, 3' exonuclease activity in the In‑Fusion enzyme mix requires terminal 3' OH groups.
  • Use our online Molar Ratio Calculator to calculate specific insert to vector quantities based on molar ratios, insert length (bp), and vector length (bp).

Can I modify the length of the homologous overlap? Will a longer overlap improve In‑Fusion Cloning efficiency?

Current In‑Fusion Cloning reaction conditions favor a 15-bp homologous overlap. We do not recommend using overlaps shorter than 12 bp or longer than 21 bp.

Will cloning efficiency increase if I use a longer incubation time for the In‑Fusion Cloning reaction?

No, an increase in the In‑Fusion reaction time is not recommended. It may generate uneven single-stranded regions at the ends of the cloning insert and vector, resulting in inefficient annealing of the homologous overlaps, thus reducing cloning efficiency.

Can I use TOP10 cells for In‑Fusion Cloning?

TOP10 cells or their derivatives (e.g., ccdB Survival 2T1R E. coli), and related strains (e.g., DH10B, MC1061) are suboptimal for In‑Fusion cloning, resulting in a lower number of recombinant clones. This may be of particular concern if you are performing multiple-fragment cloning, or using a low-copy number vector.

We recommend using Stellar Competent Cells, which are optimized for use with In Fusion Cloning and are included in all current kits.

What bacterial strains are compatible with In‑Fusion Cloning?

In‑Fusion Cloning requires bacterial cells with competency no less than 108 cfu/µg supercoiled DNA.

  • Stellar Competent Cells (included in all current In‑Fusion Cloning kits) as well as any general purpose cloning E. coli strain should be compatible with In‑Fusion Cloning.
    • Stellar Competent Cells have been validated for cloning and amplification of large vectors (e.g., BACs, fosmids) and vectors with reiterated sequences such as Long Terminal Repeats (LTRs) in retroviral/lentiviral vectors, or Inverted Terminal Repeats (ITRs) in adenoviral vectors.
  • TOP10 cells or their derivatives (e.g., ccdB Survival 2T1R E. coli), and related strains (e.g., DH10B, MC1061) are suboptimal for In‑Fusion cloning, resulting in a lower number of recombinant clones. This may be of particular concern if you are performing multiple-fragment cloning, or using a low-copy vector.

We do not recommend transforming In‑Fusion reaction mixtures into any of the following:

  • E. coli strains lacking recA1, or endA mutations
  • E. coli strains engineered for a particular application (e.g., large scale protein expression)
  • Gram-positive bacterial strains
  • Bacterial cells carrying nupG (deoR) mutations

Note: If it is absolutely necessary to use a particular bacterial strain not validated for In‑Fusion Cloning, a 1:5 dilution of the reaction mix may increase transformation efficiency.

Can I transform In‑Fusion Cloning reaction mixtures in amounts larger than what is recommended in the user manual?

We do not recommend this. Transforming the reaction mixture in an amount larger than what is stated in the user manual may be toxic to your cells. For transformation of the In‑Fusion Cloning reaction, use 2.5 µl of undiluted, unpurified reaction mix per 50 µl Stellar Competent Cells.

(Optional) For larger transformation volumes, 5.0 µl of undiluted, unpurified reaction mix can be transformed per 100 µl Stellar Comptent Cells.

In an In‑Fusion Cloning reaction, how many colonies should I expect from the negative control?

The negative control provided with the kit typically produces fewer than 5% blue colonies; the number of white colonies produced varies slightly depending on the strain. In general, fewer than 5% of the white colonies on an experimental plate contain background. It has been our observation that ≥95% of the colonies on experimental plates are correct. This speaks to In‑Fusion technology’s high level of cloning efficiency, i.e., the percentage of correct colonies recovered regardless of the total number of transformed colonies present.

Can I use electroporation to transform the In‑Fusion Cloning reaction mix?

1 µl of 1:5 diluted In‑Fusion Cloning reaction mix can be electroporated into 50 µl of electrocompetent bacterial cells.

How can I ensure transformation efficiency and overall cloning efficiency?

In‑Fusion HD Cloning Plus kits are all-in-one solutions that maintain high transformation efficiency and also provide the highest-possible level of cloning efficiency. While high transformation efficiency allows for a large number of transformed colonies, high cloning efficiency speaks to accuracy—ensuring that over 95% of transformants are correct, thus reducing the amount of time necessary to screen colonies.

  • The primers must be of good quality to ensure the sequence of the homologous region is correct, allowing the cloning reaction to proceed efficiently and accurately.
  • Clean PCR fragments are key for successful cloning. We recommend NucleoSpin Gel and PCR Clean‑Up for your purification, which is included in In‑Fusion HD Cloning Plus kits.
  • For cloning efficiency, it is important that the PCR fragment be purified away from dNTPs and PCR primers after amplification.
  • Use highly competent E. coli cells that have a transformation efficiency greater than 108 cfu/µg supercoiled DNA. Most homemade competent cells are not competent enough, especially if these cells are stored before use. We recommend Stellar Competent Cells, which are optimized for use with In‑Fusion Cloning and are included in all current kits.

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