#InFusion cloning is one of the fastest among the currently available ones, and is also widely used because of its reliability. It has several advantages over the #Gateway, #GoldenGateway and #classicalcloning techniques as is discussed later.
Principle
The In-Fusion cloning relies upon homology-based recombination between the vector and the insert molecules. The In-Fusion cloning utilizes a proprietary mix of enzymes (most probably #T5Exonuclease and others) to recombine insert(s) and vector with a common homologous 15bp flanking sequence at their linear ends. The nicks (if any) are repaired in vivo in the transformed bacteria.
Protocol
FIGURE: Schematic representation of the In-Fusion Cloning Protocol.
A. Insert PCR
The primer designing for #InsertPCR and #VectorPCR is very crucial for successful In-Fusion cloning. There are certain considerations here like:
a. length should be 18-25 nucleotides and #GCcontent must be 40-60%.
b. 3' end of primers for insert PCR should be gene-specific and for vector PCR must be vector specific.
c. Tm should be between 58-65C and difference of Tm between Forward and Reverse primers must be <=4C.
d. The last 5 nucleotides at the 3' ends should contain no more than two G/C.
After PCR, #GelExtraction of the fragment and quantification should be done.
B. Vector PCR
Vector PCR products should be long migrated in the gel to identify the correct linear band to cut and gel extract. After gel extraction it is recommended to do a #DpnI digestion step at 37C to get rid of any circular plasmid.
C. In-Fusion Reaction
The In-Fusion Reaction is simple, just add calculated amount of insert and vector, #5XInFusionMix and water (if needed) to make up a reaction volume of 10uL. The reaction is recommended to be incubated at 50C for 15mins and must immediately be placed on ice.
D. Transformation
Transform 2.5uL of the In-Fusion reaction into 50uL of highly competent E. coli cells of your choice. Follow your standard transformation protocol, plate the cells with appropriate antibiotic and incubate at 37C overnight.
E. Colony PCR/ Sequencing
Among the colonies >95% are expected to be transformants. This can be validated through colony PCR with one vector-specific and one insert-specific primer. Colony sequencing may also be done with those sets of primers.
Merits
The cloning reaction is completed in 15 mins (!)
This can be used to clone multiple DNA fragments simultaneously in a single tube, with proper #flankingsequences though!
No restrictions, clone any insert into any vector!
There is a minimum 15bp sequence constraint on the flanking sequences of the insert PCR product and the ends of the linearized vector. This shorter recombination sequence permits the overall primer length to be <40 bases, which allows for only one PCR reaction to accomplish cloning using this system.
It can efficiently and consistently clone large DNA fragments (<15kb) without requiring the cloning of multiple DNA products simultaneously.
The #ORF of interest can be directly transferred to the final expression vector of interest without an intermediate entry clone step as in the Gateway cloning scheme.
Assembled clones are seamless with no extra bases or #scars!
Demerits
There is no standard vector, you have to design primers each time and do the #PCR and/or #RestrictionDigestion beforehand.
As the repair ligation occurs in vivo, it is not possible to check the integrity of the construction before transformation.
Also as it is based on #HomologousRecombination, it does not work in any microorganisms (due to different repair systems).
Applications
Cloning any gene or DNA fragment in your vector of choice, for protein expression.
Cloning multiple DNA fragments of varying sizes.
Cloning DNA fragments or genes into shuttle vectors, also for standardizing expression hosts.
Sub-cloning.
Go get your gloves plasmidy!
For more insights:
Comments