In-Fusion® HD Cloning Kit

Easy and convenient directional cloning kit

  • Directional cloning possible at any position in any vector
  • Simply add the 15 bases of the vector's terminal sequence to the primer for amplifying the target DNA to prepare the PCR product.
  • Do not add any extra sequences
  • We have experience cloning 15 kb fragments. Simultaneous cloning of multiple fragments is also possible.
  • Widely used in laboratories around the world
Product Code Product Name Capacity Documents
639648 In-Fusion® HD Cloning Kit 10 times
639649 In-Fusion® HD Cloning Kit 50 times

Description

In-Fusion cloning system can fuse DNA fragments efficiently and quickly using In-Fusion enzymes if the terminal 15 bases of each DNA fragment are homologous. PCR fragments can be cloned into any vector in a single step without restriction enzyme digestion, blunt-ending, or ligase reactions. The In-Fusion HD Cloning Kit is a premix type, and the reaction time is only 15 minutes, with high cloning efficiency and high quality cloning.
With efficient In-Fusion cloning, appropriate clones can be obtained with minimal screening. In addition to joining two DNA fragments, it is also possible to properly link four DNA fragments in a single reaction (1). The In-Fusion cloning method is also useful for high-throughput (HTP) systems, and has been used in HTP projects at Harvard Medical School (2), Stanford Medical School (3), and Oxford University (4).

Overview of the In-Fusion reaction

Figure 1. In-Fusion cloning

  1. A 15-base sequence homologous to the terminal sequence of the vector to be used is added to the 5' end of the primer for amplifying the target gene, and the target gene is amplified by PCR.
  2. If the amplified product is a single band, perform Cloning Enhancer treatment. If multiple bands are observed, purify it from the gel and bind it to the linearized vector using In-Fusion enzyme (In-Fusion reaction: 50℃, 15 minutes). The Cloning Enhancer included in the w/Cloning Enhancer type product is a unique pretreatment reagent that allows PCR products to be used directly in In-Fusion reactions. Simply add this reagent to the reaction solution after PCR and incubate for 30 minutes, and it can be used directly in the In-Fusion reaction. This eliminates the need for a purification step for the PCR product and reduces sample loss.
  3. Transform with the In-Fusion reaction mixture and screen for the desired clones.

In-Fusion Primer Design

Designing primers for In-Fusion cloning and amplifying the target DNA fragment is basically the same as designing regular PCR primers and amplifying the target DNA fragment with PCR. The only difference is that 15 bases homologous to the terminal sequence of the vector to be used are added to the 5' end of the sequence-specific primer (see diagram below).

To design In-Fusion primers for your vector, please use the convenient online tool "In-Fusion Cloning Primer Design Tool."*The link goes to the TB USA website. The design tool has been updated to make it easier to use.*The "Primer Design tool for In-Fusion" that you have been using until now can be found Below.

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Figure 2. In-Fusion Primer Design

Primer Design Tool
Primer Design Tool

Design your primers

NEW TOOL!

Our NEW In-Fusion Cloning Primer Design Tool allows for single- or multiple-insert cloning, accommodates vector linearization by inverse PCR or restriction digest, and enables site-directed mutagenesis. Simply input the DNA sequences of your vector and insert(s), along with your linearization method to generate primers for your next cloning experiment. Easily switch to the mutagenesis option to generate primers for all of your insertion, replacement, and deletion projects.

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