In‑Fusion Cloning tips and FAQs

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.

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.

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. For multiple-insert cloning, we recommend increasing the overlap to 20 bp.

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 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.

A brief overview of the In‑Fusion Cloning protocol.

In-Fusion Snap Assembly includes liquid In‑Fusion Snap Assembly Master Mix, whereas In‑Fusion Snap Assembly EcoDry includes pre-aliquoted, lyophilized In‑Fusion Snap Assembly Master Mix. The EcoDry kit minimizes handling and is stored at room temperature. Both liquid and lyophilized master mixes have a cloning reaction of 15 min.

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 Snap Assembly User Manual). CE is available 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 yields high-quality results.

We have 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.

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

In-Fusion Snap Assembly offers all the same benefits and uses the same streamlined protocol as In-Fusion HD Cloning, but yields higher efficiency (number of colonies) and consistency, especially with challenging projects like multiple-fragment cloning.

No. While the In-Fusion HD bundles include CloneAmp HiFi PCR Premix and In-Fusion Snap Assembly bundles include PrimeSTAR Max PCR Premix, both enzymes provide exceptionally high fidelity and will give you the same trusted results.

In-Fusion Snap Assembly produces more colonies than NEBuilder HiFi for most cloning experiments (e.g., single-insert cloning, large-vector cloning, and site-directed mutagenesis) and always yields a cloning accuracy >90%. For multiple-insert cloning, In-Fusion Snap Assembly only requires a 15-minute incubation, compared with up to 60 minutes for NEBuilder HiFi.

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. For multiple-insert cloning, we recommend increasing homology to 20 nt. Figures 2 and 3 in the In‑Fusion Snap Assembly User Manual or In‑Fusion Snap Assembly EcoDry User Manual provide detailed examples.

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.

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). For multiple-insert cloning, we recommend increasing the homology to 20 nt. 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.

Current In‑Fusion reaction conditions favor 15 bp of homologous overlap for single-insert cloning, and 20 bp of homologous overlap for multiple-insert cloning. We do not recommend using overlaps shorter than 12 bp or longer than 21 bp.

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).

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

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:

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 Snap Assembly User Manual or In‑Fusion Snap Assembly EcoDry 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).

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:

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.

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.

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.

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

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

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 PrimeSTAR Max DNA Polymerase (supplied with some In‑Fusion Snap Assembly bundles). 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.

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

Yes—we have successfully tested multiple-fragment cloning with up to five inserts (see the 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.) 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.

In‑Fusion Cloning 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.

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):

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 nonspecific 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 nonspecific background, you can use the Quick In‑Fusion Cloning Protocol provided in Appendix A of the In-Fusion HD Cloning Kit 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.

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.

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

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.

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, the integrity of the linearized vector termini is essential. We recommend using high-quality restriction enzymes and performing digests over several hours. However, overnight restriction digests are not advisable.
  • Dephosphorylation of the vector termini is not required; the vector will not recircularize 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.
  • 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 PrimeSTAR Max DNA Polymerase (supplied with some In‑Fusion Snap Assembly bundles). 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.

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.

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.)

No, dephosphorylation of the vector termini is neither required nor recommended for 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.)

Yes—our 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).

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.

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

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.

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 generation of the 15-bp overlaps at the termini.

Yes, In‑Fusion Cloning allows single or multiple base changes, deletions, and insertions. For details, please see the Mutagenesis with In‑Fusion Cloning tech note.

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.

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 Cloning master mix 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.

In‑Fusion Cloning 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).

Current In‑Fusion Cloning reaction conditions favor a 15-bp homologous overlap for single-insert cloning, and a 20-bp homologous overlap for multiple-insert cloning. We do not recommend using overlaps shorter than 12 bp or longer than 21 bp.

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.

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.

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-number 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.

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 of Stellar Competent Cells.

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

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.

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

In‑Fusion Cloning is an all-in-one solution that maintains high transformation efficiency and also provides 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 some In‑Fusion Snap Assembly bundles.
• 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 bundles.

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. We recommend increasing the overlap to 20 bp of homology for multiple-insert cloning.

• 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 homologous overlap.
• 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 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 the 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.

In‑Fusion Snap Assembly master mixes are available in a lyophilized (EcoDry) or liquid format. Each format also contains reagents for control experiments (linearized vector, 2-kb 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, are you interested in other products to increase your cloning efficiency (Stellar competent cells, PCR product purification kits, and PCR enzymes)? The table below illustrates the differences between the lyophilized and liquid kit formats.

Lyophilized versus liquid kit formats
Feature	In‑Fusion Snap Assembly EcoDry	In‑Fusion Snap Assembly (liquid)
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
15-minute reaction time

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.

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 Cloning, was transformed in chemically competent bacteria, allowing the rescue of the recombinant vector. It was not used in the actual cosmid packaging reaction.

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 recircularize 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.
  • 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 PrimeSTAR Max DNA Polymerase (supplied with some In‑Fusion Snap Assembly bundles). 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.  

• 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.

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.

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.

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

Seamless cloning is not suitable for small inserts of less than 50 bp. This is because of the exonuclease activity removing a 15–30 bp region at both ends, which may affect a significant percentage of the insert sequence.

We recommend using a traditional, ligase-based cloning method for inserts smaller than 50 bp.

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).

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.

 

In‑Fusion Cloning 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.

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.
  
  
  • We recommend increasing homology to 20 bp for multiple-insert cloning.
  • 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.

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.

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
  • Prelinearized vectors
  • Insert-specific primers designed by the user
  • Insert-specific primers generated by the Primer Design Tool
• 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.

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).

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.

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.

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):

In‑Fusion Cloning allows single or multiple base changes, deletions, and insertions. For details, please see our Mutagenesis with In‑Fusion Cloning tech note.

In‑Fusion Cloning 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).

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).

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.

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.

Current In‑Fusion Cloning reaction conditions favor a 15-bp or 20-bp homologous overlap for single-insert or multiple-insert cloning, respectively. We do not recommend using overlaps shorter than 12 bp or longer than 21 bp.

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 PrimeStar Max DNA Polymerase (supplied with some In-Fusion Snap Assembly bundles). 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.

• 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 T_m 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 T_m 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.

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.

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

• We recommend Stellar Competent Cells (included in all In‑Fusion Snap Assembly bundles). 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.

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.

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 of 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.

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.).

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 listed under "Documents" in the "Details" section of the product table listing for a given vector on the In-Fusion Ready green and red fluorescent protein vectors product pages.
   
   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.

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 Snap Assembly User Manual and the In‑Fusion Snap Assembly EcoDry 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.
   • 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 Snap Assembly User Manual or the In‑Fusion Snap Assembly EcoDry User Manual for In‑Fusion reaction and transformation instructions.

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.

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.

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

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.)

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.

In‑Fusion Cloning 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.

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 PrimeSTAR Max DNA Polymerase. 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.

• 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 recircularization 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 Mutagenesis with In‑Fusion Cloning tech note.

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.

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.

• 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 coexpressed 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.
• We offer Tet-inducible miRNA expression systems and vectors with either a red or green fluorescent protein marker.