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Products >  Cloning_and_Competent_Cells >  Cloning_Resources >  Cloning_Tips >  In-Fusion_Cloning

In‑Fusion Cloning Tips

In-Fusion Cloning is a highly efficient, ligation-independent 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.

The following information applies 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

Planning your experiment

Successful In‑Fusion Cloning reactions require 15-bp homologous overlaps at the termini of the cloning insert and linearized vector, or adjacent inserts if multiple inserts are to be joined simultaneously.

  • These overlaps can be generated by PCR amplification or oligo synthesis of either of the cloning fragments.
  • 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.

A 3' exonuclease in the In‑Fusion enzyme mix generates 15-nt single-stranded 5' overhangs at the termini of the cloning insert and linearized 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.

Choosing a kit format

In‑Fusion HD Cloning Plus Kits are available in a lyophilized (EcoDry) or liquid format. Each of these kits contain CloneAmp HiFi PCR Premix, In‑Fusion HD enzyme premix, and reagents for control experiments (linearized vector, 2kb insert).

There are two decisions to make when deciding on a kit format. First, do you want a lyophilized or liquid kit format and second, what PCR product purification method is suitable for your amplified DNA insert? The table below illustrates the differences between the lyophilized and liquid kit formats. After you make this decision, continue reading to learn the different options for PCR product purification.

Lyophilized versus liquid kit formats
Feature In‑Fusion HD EcoDry Cloning Plus In‑Fusion HD Cloning Plus
Pre-aliquoted, lyophilized components that minimize handling errors
Room temperature storage, saving freezer space and eliminating freeze-thaw cycles
Ability to customize: reaction volumes and/or plasticware
30-minute reaction time
15-minute reaction time

Purification of PCR products:

It is important to purify PCR products and vectors prior to combining in the cloning reaction. Both In‑Fusion HD EcoDry Cloning Plus and In‑Fusion HD Cloning Plus kits include a NucleoSpin Gel and PCR Clean-Up kit for purification of PCR products. These kits are suitable for PCR and gel extraction in situations where PCR produces more than one product.

In‑Fusion HD Cloning Plus CE contains all the kit components provided in other In‑Fusion Cloning Plus kits, but Cloning Enhancer (CE) is included for treatment of PCR products in place of NucleoSpin Gel and PCR Clean-Up. 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. 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 (HTP) applications that employ highly optimized PCR cycling conditions and primers that generate clean products of the expected size.

Vectors

Compatible vectors

Any linear vector can be used for In‑Fusion Cloning, regardless of size, composition, or available restriction site(s). The cloning reaction is followed by the rescue of a circular recombinant construct in E. coli, but please note that circular vectors are not compatible with the cloning reaction itself. Additionally, In‑Fusion Cloning does not allow the assembly of covalently linked linear DNA molecules.

Vector size

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, cosmids at 46 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.)

*

The 46-kb cosmid vector, assembled by In-Fusion HD Cloning, was transformed in chemically competent bacteria, allowing the rescue of the recombinant vector. It was not used in the actual cosmid packaging reaction.

Vector linearization and purification

Linearization options include:

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

    • 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.

Inserts

Insert sources

The following types of inserts are compatible with In‑Fusion Cloning:

  • PCR-amplified DNA fragments carrying overhangs complementary with the termini of the adjacent DNA fragment(s), synthetic oligos, or linear vector.
  • DNA fragments generated by restriction digest with one or more restriction enzymes. In this instance, the required 15-bp homology must be carried by the adjacent DNA fragment(s), synthetic oligos, or linear vector.
  • Synthetic oligonucleotides (≥50 bp) with 15-bp homology to the termini of adjacent fragments or the linearized vector. High-quality, non-phosphorylated oligonucleotides purified by desalting are compatible with In‑Fusion Cloning. Gel or HPLC purification of oligonucleotides is not required.

Large inserts

In-Fusion 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).

Small inserts

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, the 3' exonuclease activity in the In‑Fusion enzyme mix requires terminal 3' OH groups.

Multiple inserts

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

Primer Design

PCR primers compatible with In‑Fusion Cloning

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

  • 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.
    • Note: When a vector has been linearized via restriction digest, the 5' overhang of a restriction site is included in the 15 nt of homology, while the 3' overhang of a restriction site is excluded from the count of the 15 nt of homology.
  • (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.

Generating homologous overlaps of DNA fragments

15-bp homologous overlaps between cloning termini facilitate all In‑Fusion Cloning reactions. They can be generated in the following ways:

  • PCR amplification of a cloning insert using PCR primers carrying 15-nt 5' overhangs that are homologous to the termini of the linearized vector or adjacent insert
  • PCR linearization of the destination vector, using PCR primers carrying 15-nt 5' overhangs homologous with the cloning insert(s)
  • Oligonucleotide synthesis, generating 15-nt 5' end overhangs homologous with the termini of the linearized vector or adjacent insert. High-quality, non-phosphorylated oligonucleotides purified by desalting are compatible with In‑Fusion Cloning. Gel or HPLC purification of oligonucleotides is not required.

Primer design tools

Instructions for designing In‑Fusion PCR primers are included in all In‑Fusion Cloning user manuals. Several tools are also available to help with the process.

  • Our online Primer Design Tool facilitates primer design for In‑Fusion Cloning, and is compatible with Mozilla Firefox or Google Chrome web browsers. (Internet Explorer is not compatible with the Primer Design Tool.) Specific cloning conditions supported are as follows:
    • Single-fragment cloning
    • Multiple-fragment cloning
    • Vectors linearized by restriction digest
    • Vectors linearized by inverse PCR
    • Pre-linearized vectors
    • Insert-specific primers designed by the user
    • Insert-specific primers generated by the Primer Design Tool
  • Step-by-step tutorials for the Primer Design Tool are also available in the Cloning Resources section of our website.
  • The current version of the Primer Design Tool does not allow adjustment for translational reading frame continuity, which should be manually designed by the user.
  • 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.

Miscellaneous Options

Restriction site preservation

Primer design lets you easily preserve or eliminate the restriction sites used to linearize the cloning vector. In order to maintain restriction sites at cloning junctions, nucleotides can be added to the PCR primers between the template-specific portion and the 15-nt homologous overlap.

The online Primer Design Tool offers the option to preserve the restriction site(s). (The Primer Design Tool is compatible with Mozilla Firefox and Google Chrome web browsers, but not with Internet Explorer.)

Translational reading frames

In‑Fusion Cloning makes it possible to seamlessly clone your gene of interest in the same translational reading frame as a desired tag (e.g., fluorescent protein, Myc, HA, etc.). Specifics in the design of In‑Fusion PCR primers facilitate this application, allowing you to maintain reading frame continuity in the recombinant vector. Two options for doing so are below:

  • Adjusting the length of the template-specific (gene-specific) portion of the In‑Fusion PCR primer
  • Adding nucleotides between the template-specific portion and the 15-nt homologous overlap portion of the In‑Fusion PCR primer

Please note that the current version of our online Primer Design Tool does not allow adjustment for translational reading frame continuity. 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.

Inserting external nucleotide sequences

Small external nucleotide sequences (e.g., small tags, Kozak sequences, restriction sites, cleavage 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.

Splitting the 15-nt homologous overlap

The homologous 15-nt overlap can be split between two adjacent DNA fragments. It can only be split between an insert and vector 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

Site-directed mutagenesis

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

General Guidelines

Molar ratios

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).

Control reactions

Always perform a control In‑Fusion Cloning reaction using the control vector (linearized pUC19) and control insert provided in each kit. If your experiment produces unexpected results, the control reaction can help you to determine where to start troubleshooting.

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 bacterial strain used for transformation. 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.

Incubation time

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.

Location of homologous overlaps

The 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 locus, regardless of available restriction sites, thus enabling the generation of the 15-bp overlaps at the termini at a particular position.

Length of homologous overlaps

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.

PCR Requirements

Compatible polymerases

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.

Tips for amplification with In‑Fusion PCR primers

  • The template-specific 3' end of the In‑Fusion PCR primer should be 18–25 nt long, in order to ensure template amplification.
  • To determine the Tm of In‑Fusion PCR primers, use independent software for PCR primer analysis, such as OligoAnalyzer 3.1 from IDT Technologies. Use standard Mg2+, Na+, and dNTP concentrations usually recommended for PCR.
  • For optimal amplification, perform the initial 3–5 PCR cycles using the annealing temperature compatible with just the 3' template-specific portion of the In‑Fusion PCR primer. The remaining PCR cycles should use an annealing temperature compatible with the Tm of the entire primer.
  • If you experience inefficient PCR amplification, it may be necessary to re-design the In‑Fusion PCR primers, by either extending or repositioning the template-specific 3' end.

Compatible PCR purification methods

PCR-amplified DNA must be purified prior to the In‑Fusion Cloning reaction. This can be accomplished with one of the following options:

  • 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 high-throughput (HTP) applications that employ highly optimized PCR cycling conditions and primers that generate 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.

Transformation in E. coli

Cell competency

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

  • We recommend Stellar Competent Cells (included in all current In‑Fusion Cloning kits). Any general-purpose cloning E. coli strain should be compatible with In‑Fusion Cloning as well.
    • 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 number vector.

Strains not recommended for In‑Fusion Cloning

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.

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.

Optimal transformation amounts

For transformation of chemically competent bacterial cells (e.g., Stellar Competent Cells), use 2.5 µl of undiluted In‑Fusion Cloning reaction mix per 50 µl of cells.

For transformation of electrocompetent cells, use 1 µl of 1:5 diluted In‑Fusion Cloning reaction mix per 50 µl of cells.

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

We advise against transforming the reaction mix in amounts larger than those stated above, as it may be toxic to your cells.

Vector and insert properties related to transformation efficiency

Some vectors or inserts may contain reiterated sequences (e.g., retroviral or lentiviral LTRs, adenoviral ITRs, etc.). When working with such vectors, it may be necessary to optimize bacterial transformation to prevent rearrangements within the recombinant construct and increase its stability during rescue and amplification in E. coli. Follow heat shock with revival of the bacteria at 25–30°C while shaking at 120–160 rpm, and varying the selective antibiotic concentration and/or growth temperature of the plated transformation culture (e.g., 25°C, 30°C, etc.).

Selected Applications

Cloning a gene of interest in-frame with a fluorescent protein (easy protocol)

  1. Use In‑Fusion Ready Fluorescent Protein Vectors
  2. Design In‑Fusion PCR primers according to the following resources: pAcGFP1-N In‑Fusion Ready vector information, pAcGFP1-C In‑Fusion Ready vector information, or the vector information on the Documents tab of the In-Fusion Ready Fluorescent Protein Vectors product page.

    Note: To preserve translational reading frame continuity, your gene of interest should not contain a STOP codon if inserted upstream of the fluorescent protein. If the gene of interest is downstream of the fluorescent protein, it may retain its own STOP codon, but the fluorescent protein should not.

  3. Amplify your gene of interest using the In‑Fusion PCR primers designed in Step 2.
  4. Purify the amplified gene of interest using NucleoSpin Gel and PCR Clean-Up or Cloning Enhancer.
  5. Follow the In‑Fusion Ready Vector Cloning Protocol-At-A-Glance for In‑Fusion reaction and transformation instructions.

Cloning a gene of interest in-frame with a fluorescent protein (alternative protocol)

  1. Choose any vector carrying a fluorescent protein. Linearize your vector either by restriction digest or inverse PCR.
  2. Purify the linearized vector by gel extraction to ensure the isolation of only linear vector molecules.
  3. Design In‑Fusion PCR primers using the online Primer Design Tool. (The Primer Design Tool is compatible with Mozilla Firefox or Google Chrome web browsers, but not with Internet Explorer.)
    • Alternatively, instructions for In‑Fusion primer design are described in the In‑Fusion HD Cloning Kit User Manual and In‑Fusion HD EcoDry Cloning Kit User Manual.
    • Regardless of design method, we recommend SnapGene Viewer for in silico assembly of your recombinant construct and easy visualization of the locations of In‑Fusion primers in the sense and antisense strands.
    • Notes: To preserve translational reading frame continuity, your gene of interest should not contain a STOP codon if inserted upstream of the fluorescent protein. If the gene of interest is downstream of the fluorescent protein, it may retain its own STOP codon, but the fluorescent protein should not.
    • If necessary, you can also adjust the reading frame continuity by inserting extra nucleotides between the template/gene-specific portion and the 15-nt homologous portion of the In‑Fusion PCR primers, as illustrated in the table below. (The current version of our Primer Design Tool does not allow for this type of adjustment, and requires manual design by the user.)

    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
  4. Amplify your gene of interest using the In‑Fusion PCR primers designed in Step 3.
  5. Purify the amplified gene of interest using NucleoSpin Gel and PCR Clean-Up or Cloning Enhancer.
  6. Follow the In‑Fusion HD Cloning Kit User Manual or In‑Fusion HD EcoDry Cloning Kit User Manual for In‑Fusion reaction and transformation instructions.

Compatibility with large vectors

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.)

Compatibility with large inserts

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).

Multiple-fragment cloning

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, or split between them.

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

Mutagenesis

In‑Fusion Cloning allows single or multiple base changes, deletions, and insertions through the use of inverse PCR. Please note this application relies on high-fidelity PCR. It is essential to use a PCR polymerase with high proofreading activity, such as 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, and 15 kb of Lambda DNA, 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.

  • Each PCR primer directs DNA synthesis in the opposite orientation of the other on a circular vector template.
  • The 3' ends of the forward and reverse PCR primers are 18–25 nt that are complementary to the template, ensuring efficient and specific amplification.
  • Mutations are incorporated within the homologous 15-nt overlap located at the 5' ends of the forward and reverse PCR primers. (This homologous overlap is required for the re-circularization of the mutated vector.)
    • Single or multiple base changes, deletions, or insertions can be introduced in a single In‑Fusion reaction.
    • A larger deletion of any desirable length can also be introduced by positioning the 3' ends of the forward and reverse primers at the border sites of a deletion, with homologous overhangs carried by the 5' end of either of the primers.
  • The resulting inverse PCR will generate a linear double-stranded vector with 5' and 3' ends complementary to each other, and carrying the 15-nt homologous overlap. (This overlap will be joined through the In‑Fusion reaction and recovery in E. coli, thus generating a mutated vector.)
  • Vectors amplified with inverse PCR must be treated with Cloning Enhancer to destroy the parental vector. Additional purification by preparative gel electrophoresis may be required to ensure isolation of the linearized vector from PCR byproducts and possible remnants of the parental circular vector.

For additional details, please see our prerecorded mutagenesis webinar and/or the Mutagenesis with In‑Fusion HD Cloning Plus tech note.

Cloning shRNA (small hairpin RNA)

An shRNA double-stranded DNA oligonucleotide (≥50 bp) can be cloned via In‑Fusion technology into a linearized shRNA expression vector.

  • 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 the oligo length, the optimal ratio must be determined empirically.
  • High-quality, non-phosphorylated oligonucleotides purified by desalting are compatible with In‑Fusion Cloning. However, 3' exonuclease activity in the In‑Fusion enzyme mix requires terminal 3' OH groups.
  • Clontech offers a wide variety of shRNA expression vectors. These vectors also allow shRNA cloning via restriction digest and ligation, using shRNA oligos with restriction site overhangs (generated by the Online shRNA Sequence Designer).

Note: Not all antisense oligonucleotides designed and tested for direct cell transfection, such as siRNAs, will be equally efficient when expressed as an shRNA from a vector. It is usually recommended to redesign the siRNA oligo for expression as an shRNA with various orientations of the target sequence as a sense or antisense strand.

  • For efficient knockdown, at least four different shRNA constructs are typically designed and tested first in transient transfection (using easy-to-transfect cells, if applicable), prior to establishing a stable cell line or running in vivo experiments.
  • In order to distinguish recombinant shRNA vectors, a diagnostic restriction site (MluI) can be inserted into the shRNA oligo downstream from the RNA Polymerase III Termination Signal.

Cloning a microRNA (miRNA) precursor

  • Sequences for microRNA precursors and flanking genomic DNA can be obtained from a number of public databases, including GenBank and EMBL-Bank. The UCSC Genomic Bioniformatics Site hosts an easy-to-navigate genomic database which tracks miRNAs. The Sanger Institute hosts miRBase, a compilation of known miRNA sequences.
  • 100–300 bp of DNA flanking the miRNA precursor is amplified from genomic DNA for cloning into the 3' UTR of a fluorescent protein, carried by an miRNA expression vector. The flanking DNA ensures efficient processing by Drosha.
  • For In‑Fusion Cloning, the miRNA precursor (100–300 bp) is PCR amplified, incorporating 15-bp overhangs homologous to the termini of the miRNA expression vector. The vector should be linearized at the fluorescent protein's 3' UTR. The suggested miRNA precursor to vector molar ratio for the cloning reaction is 3–5:1, depending on the precursor length. Optimal molar ratios must be determined empirically.
  • In the cell, the miRNA precursor is co-expressed with the fluorescent protein (as described in Figure 2 of this tech note), allowing both of the following:
    • Expression of the fluorescent protein, resulting in fluorescent cell labeling.
    • miRNA precursor processing, resulting in targeted gene knockdown in fluorescently labeled cells.
  • Clontech offers Tet-inducible miRNA expression systems and vectors with either a red or green fluorescent protein marker.

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