User:Jarle Pahr/Cloning

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5 Tips on Vector Preparation for Gene Cloning:

Gateway cloning:


Inverse Fusion PCR cloning:

Blunt end ligation:

In-Fusion Cloning primer design tool:


Commercial kits

Electra Vector system:


Conventional (restriction-ligation) cloning

Ligation Independent Cloning (LIC)

Adding RecA may improve yield?


If no colonies are achieved, try "quick and dirty" cloning:

A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production:

A Family of LIC Vectors for High-Throughput Cloning and Purification of Proteins:

Gibson Assembly

From J5 manual, "The SLIC, Gibson, CPEC and SLiCE assembly methods" ( :

"SLIC, Gibson, CPEC, and SLiCE are related methods that offer standardized, scarless, (largely) sequence-independent, multi-part DNA assembly".

Also: "Despite their differences in implementation, SLIC, Gibson, CPEC, and SLiCE assembly methods all start with the same starting materials and result in the same final products"

A guide to Gibson Assembly:

See also

NEBs NEBuilder software for Gibson assembly can be used to design primers for Gibson assembly and SLIC:

In the NEBuilder, the overlaps are designed to achieve a minimum Tm value of 48°C.

Hetero-stagger PCR and Enzyme-Free cloning

Articles related to "hetero-stagger PCR" and "enzyme free cloning" (Conceptually equal to PIPE/iPCR):

Author(s)/year Comment Reference
Liu 1996: Hetero-stagger cloning: efficient and rapid cloning of PCR productsGeneration of related PCR products by using two sets of primers, identical except for a 3bp GGG addition at 5' end in one set. After mixing the PCR products, denaturing and reannealing, a mixed product with CCC overhang is produced, which can be cloned by ligation to a vector with a GGG overhang.
Tillett and Neilan 1999: Enzyme-free cloning: a rapid method to clone PCR products independent of vector restriction enzyme sitesBuilds on Liu 1996. Both insert and vector is prepared by hetero-stagger PCR and mixed before undergoing denaturation/annealing to give the desired construct. Article is reproduced in part in [PCR Cloning Protocols, 2nd edition.
Shih, Y.P., Kung, W.M., Chen, J.C., Yeh, C.H., Wang, A.H. and Wang, T.F. (2002) High-throughput screening of soluble recombinant proteinsApplication of "sticky end PCR method" (hetero-stagger PCR) to High-throughput (HTP) cloning.
de Jong, R.N., Daniëls, M.A., Kaptein, R. and Folkers,G.E. (2007) Enzyme Free Cloning for high throughput gene cloning and expression.Combines preparation of insert by hetero-stagger PCR amplification of preparation of vector by exonuclease chewback, applied to HTP cloning.

de Jong et al. 2007 comments: "An ideal cloning method should require small amounts of insert and vector DNA, and be cheap, effective, easily automatable and insensitive to variations in DNA concentrations [24]. Ligation independent cloning [15] meets most of these criteria, but we observed substantial variation in cloning efficiency. Parameters influencing efficiency are the amount of PCR product, purity of the PCR product, exact temperature of T4 DNA polymerase treatment and T4 DNA polymerase activity differences, caused by enzyme batch variations and activity loss over time (unpublished results). As a result, PCR products can be under- or overtreated by the exonuclease activity of T4 DNA polymerase, which can significantly influence the cloning efficiency."

See also:


Enzyme free cloning for high throughput gene cloning and expression.

Note that by cutting a vector with two restriction enzymes and using SLIC to insert a sequence, the restriction sites may be destroyed. Always consider the resulting sequences carefully.

incomplete PCR (iPCR):

Articles related to LIC/SLIC and homologous recombination:

Author(s)/year Year Comment Reference
Aslandis & Jong 1990Introduction of LIC
Bubeck et al. 1993:Rapid cloning by homologous recombination in vivo
Hsiao 1993 Exonuclease III induced ligase-free directional subcloning of PCR products
C Aslanidis, P J de Jong and G Schmitz 1994Study on minimal length requirement of SLIC overlaps. Lowest overlap length yielding transformants was 10nt.
Lisa D Cabrita, Weiwen Dai and Stephen P Bottomley 2005A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production
Li & Elledge 2007"Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC."
2012One-step SLIC

According to Olieric 2010 (Supplementary material), "The critical parameter for success is the sequence of the overlap; some constructs work amazingly well, while others do not". The cause could plausibly be stable secondary structures? Stable secondary structures in the single-stranded DNA will compete with annealing of the fragments.

Quote from Li and Elledge 2007:

"Excision by the proofreading exonuclease of T4 DNA polymerase has proven to be the most reproducible and easiest to manipulate method for generating 5¢ overhangs. Although much less efficient, iPCR also gives substantial stimulation of transformation. This might be sufficient for routine subcloning purposes, although there is likely to be more variability depending on the completeness of the PCR synthesis"

Protocols for LIC/SLIC and related methods:

Author(s) Hsiao 1993 Aslandis & Jong 1990
Method: With RecA Without RecA With iPCR Exonuclease IIILIC
Step 1
Step 2
Step 3

Two-step SLIC has the advantage that larger amounts of vector can be prepared beforehand, and used in several annealing reactions.

SEFC/Co-transformation cloning

Mix 5 μl of purified PCR product (100–300 ng) and 1 μl (50 ng) of the appropriately linearized vector, having 15+ bp homologous overlap, before transformation.




Quote from the above: "Perhaps the most significant limitation of the Golden Gate method is that it is less sequence-independent than SLIC/Gibson/CPEC/SLiCE, in the sense that, like BioBrick assembly, the selected type IIs recognition site (e.g. BsaI) should be absent from the internal portions of all of the DNA fragments to be assembled" From the same J5 website (

"Since there are no (or very few) re-amplifications of a given template sequence, PCR-derived mutations are not propagated to the same extent as one would anticipate for standard SOEing reactions. Like SLIC and Gibson assembly, CPEC is standardized, scar-less, and largely sequence-independent."

RF cloning



  • PCR procedure possibly giving higher chance of mutations

Molar ratio calculator:

RF cloning can be used to insert a sequence into a plasmid without removing any of the original plasmid sequence, or to replace a portion of a plasmid with a new sequence. RF cloning is accomplished using two PCR steps. In the first PCR, the sequence to be inserted is amplified (or synthesized) to give a product with the insert sequence flanked by sequences homologous to the plasmid. In the second PCR, the product of the first step is used as a "megaprimer" To insert a sequence into a plasmid without removing any of the original plasmid sequence, the plasmid-binding regions of the megaprimer should bind to the plasmid directly adjacent of each other, as any sequence in the plasmid between the two primer binding sites will be lost. To replace a portion of a plasmid, the plasmid-binding flanking sequences of the megaprimer must be designed to bind such that they flank the area to be replaced. If RF cloning is used to replace a portion of a plasmid, the new sequence should be about as long as the sequence it is replacing.

Articles related to RF cloning:

Author(s)/year Year Comment Reference
van den Ent & Löve 2006:RF cloning: a restriction-free method for inserting target genes into plasmidsIntroduction of RF cloning.
Unger et al. 2010: Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression
Bond & Naus 2012: an online tool for the design of restriction-free cloning projects
Erijman et al. 2011: Transfer-PCR (TPCR): A highway for DNA cloning and protein engineering

Topo cloning

Cloning methods

Method Summary Advantages Disadvantages Time (for assembly from prepared vector and insert) Experiences Reference
Conventional (restriction enzymes)Restriction digestion and ligation Predictable. With respect to vector, sequence dependence is limited to a few bp, thus cloning is robust with respect to mutations in the vector. Time-consuming. Requires restriction digestion of insert and vector followed by ligation. Requires suitable restriction sites in vector. Cloning is subject to sequence limitations in insert (must not contain restriction sites used for cloning). Restriction reaction can be spoiled by contaminating DNA flanked by restriction sites used. From 3 h to overnight
Blunt-end ligationLigation of blunt-ended fragmentsFlexible, does not require specific restriction sites.10-100x less efficients than cohesive-end ligation. Requires screening of colonies (insertion is non-directional). From 3 h to overnight
TA cloningLigation of PCR products with '3 A overhangs into vector with G' overhangs. No enzymatic treatment of insert PCR product.Non-directional
LICAnnealing of fragments with standardized homologous overlaps lackign one nucleotideCan generate specific overhangs by exonuclease incubation in presence of a single dNTP. Standardized overlaps gives higher predictability(?).Requires specific sequence in vector? Aslanidis & Jong 1990
SLIC (one-step) Annealing of fragments with varying homologous overlaps Fast Insert should be at least 250 bp. More secondary structures might be present when incubating at room temperature, compared to two-step SLIC and Gibson assembly. ~30 min
SLIC (two-step) Annealing of fragments with Homologous overlaps Fast. Assembly is carried out at higher temperature than one-step SLIC, meaning secondary structures less problematic? Slower than one-step SLIC

iPCR Annealing of vector and incomplete PCR fragments with homologous overlap No enzymatic treatment of insert PCR product. Low efficiency ~ 1 h
PIPEAmplification of vector and insert by PCR yields incomplete PCR (iPCR) products which can anneal to each other. No enzymes required (except for PCR) 1,2,3,4,5
CPEC Circularization of linearized vector and insert with homologous overlap Fast Requires linearized vector ~1h (20 cycle PCR)
Gibson Assembly Annealing of fragments with homologous overlaps. Relatively fast. Assembly is carried out at higher temperature than SLIC, meaning secondary structures less problematic? More enzymes required than for SLIC.
RF cloningAddition or substitution of a sequence in a plasmid by PCR Can use uncut vector as starting material. Can be used to either replace to replace a sequence in the original plasmid with a new one, or insert a new sequence without removing any of the original plasmid sequence. If the insert shall replace a sequence, the insert and the sequence to be replaced must be of about the same size. (Anectodal. Source?) ~3h (?)
Enzyme-free cloning Another name for PIPE/iPCR 1,2,3
Sucessive Hybridization Assembly (SHA) In Vitro Assembly of Multiple DNA Fragments Using Successive Hybridization
Simple CloningPCR Assembly of insert and vector with homologous overlaps.
Seamless Enzyme-Free Cloning (SEFC)/co-transformation cloningCo-transformation of linear insert and vector fragmentsSimple, one-step co-transformation of insert and vector. No exonuclease treatment. Requires high-efficiency competent cells (>1 × 108 colony-forming units [cfu]/μg DNA) 1 2
Transfer PCRApplication of RF-cloning for PCR amplification from

an origin vector and subsequent integration of the PCR product into a recipient vector.|| || || || ||


MASTER ligation:

"clonetegration". One-Step Cloning and Chromosomal Integration of DNA:

Use of Phosphatases:

Older articles

Exonuclease III induced ligase-free directional subcloning of PCR products:

Rapid cloning by homologous recombination in vivo:

Sticky-end PCR: new method for subcloning:

Cloning strategies



Web applications

Personal tools