Seamless Recombination for Cloning

May 09, 2017

UC Davis
Biochemistry and Molecular Medicine
Graduate Student Researcher

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Quality of Results

Ease-of-Optimization

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Company:

Takara Bio

Product Name:

In-fusion HD Cloning Plus Kit

Catalog Number:

638909

We are trying to clone a plasmid by ligating our target gene sequence into a designated vector. This PCR cloning kit is unique because it uses recombination to ligate the PCR product to the vector, which avoids errors by ligating sticky ends by conventional restriction site ligation. We chose this product because it contains a high fidelity taq polymerase for PCR amplification, a recombination enzyme, as well as competent cells for transformation. Our lab uses this product very frequently because it contains all the components we need.

Experimental Design and Results Summary

Application

Cloning

Starting Material

Plasmid DNA

Protocol Overview

Designed primers were used to amplify a PCR product using the provided high fidelity taq polymerase. After purification, it is then ligated to the vector of interest with the provided 5X In-Fusion HD Enzyme Premix and only requires 15 minutes incubation at 50C. The seamed product is then transformed into the provided HST08 competent cells and plated on solid media. The key point is to pay attention to the heatshock time and temperature. Another critical step is to ensure the PCR product to vector ratio is optimized, depending on the success of amplification.

Tips

Competent cells should be aliquoted upon arrival. Each tube contains 100uL of competent cells and cannot be repeatedly freeze-thawed.

Results Summary

The cloning kit has a very high success rate, and positive controls are given to ensure that the workflow has occurred properly. Generally, after one hour incubation of the starter culture in 700uL of SOC media, a plate usually contains 100+ colonies after overnight incubation.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

The kit has all the necessary components from amplification to ligation to transformation.

The Bad

The kit is quite expensive and the 5X In-Fusion HD Enzyme Premix is the limiting factor.

The Bottom Line

This kit generally gives 100% correct positive clones. The taq polymerase is highly accurate and recombination ligation occurs very quickly, in 15 minutes. The most convenient aspect of the kit is that the entire process only takes one day to complete before colonies are obtained.

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