Molecular cloning can be an art

As with this painted representation of (surrealist!) DNA, to a certain extent, molecular cloning is an art.

Unfortunately for newcomers, or fortunately for experts, it’s required for many downstream applications including protein production and cell engineering to develop cell-based assays. ORF expression is also a major tool for understanding a protein function via overexpression study.

In short, to develop the potential of cell biology research, it’s often a question of adopting a “smart molecular biology” approach. Along the way though, it’s unlikely that you’ll be able to avoid using cloning vectors and other related molecular cloning tools.

However… luckily, fast and simple cloning is possible! Mainly thanks to the smart kits and systems that I’ll explain more about in this post. And for your complete peace of mind, there are also ORF expression clones…

Fast fusion

The ligation step to join a PCR insert and linearised plasmid is often laborious and time consuming. It’s a tricky step, which requires optimisations. In order to simplify it, there is an alternative enzyme called Fast-Fusion™ clonase. Base on homology arms, it works like a recombinase as shown below.

clonase enzyme

Fast-Fusion clonase inserts the PCR amplicon into the vector within 15 minutes

The Fast-Fusion reveals high performances, with almost 100% cloning efficiency, on various size of inserts from 500pb to 11kpb.

Agarose gel based cloning control

Fast-Fusion cloning efficiency analysed in agarose gel

What are the advantages of Fast-Fusion™ Clonase?

  • Very fast – 15 minutes
  • Cost saving and convenient – no restriction enzyme required
  • Time saving – no digestion of the PCR insert
  • Reliable and efficient – high cloning efficiency even with long inserts
  • Compatible with complex cloning – up to 8 DNA fragments all together

Blunt-end PCR

Fast-Fusion requires adding homology sequences to the PCR insert. If you prefer to not do this, there is the option to work directly with the blunt-end PCR amplicon. Here again, no restriction enzyme is required. This innovation is called the Smart-Join Blunt-end PCR Cloning Kit, which was recently developped by GeneCopoeia. The reaction time for the key ligation step is quite reasonable, at 30 minutes. Performance depends on the insert size and is competitive as illustrated below (the one shown in red by GeneCopoeia is the one we provide).

Ligation efficiency

Positive ligation rate comparison

Gateway-based system

The EZShuttle™ LR Recombination Cloning System has the same principle as the Gateway® technology, based on recombination with Phage lambda.

Gateway alternative

Mechanism of the GeneCopoeia recombination system

Thus, based on attP, attL and attR sequence recombination, the EZShuttle system, as per the Gateway, can transfer oriented inserts from a donor vector to a receiver vector as described below.

EZshuttle cloning system

Recombination-based cloning system principle

The donor vector is the Shuttle clones, called EZShuttle Gateway Plus ORF clones, covering all the Human and Mouse ORF. The receiver vector is the pEZ expression vectors offering many choices of promoters, selection agents, and tags. A typical example is the pEZ-Lv233 that allows expression of the ORF under EF1a promoter with puromycin and eGFP under SV40 promoter. It is also a lentiviral vector, HIV type. All vectors are amplifiable.

Cloning vector

Receiver vector with EF1a promoter, puromycin and eGFP reporter under SV40

Ready-to-use clones

Despite these smart cloning systems and fast kits (Fast Fusion and Smart Joint), you might still be reluctant to invest time in molecular cloning even though it’s required for your projects. For this reason, we offer ORF directly into the expression clones.

Expression clones

ORF provided directly into expression clones

There are more than 100 pReceiver vectors corresponding to several tag possibilities and selection agents.

Tags for expression clones

List of tags possible

They also cover several cell species for the expression including Mammalian, Bacteria, Yeast and Insect cells. To make it easy, you can simply ask us, mentioning the ORF of interest, the tag if any, and the selection agent.

Expression clones

ORF expression vectors for several models

Alternatively, some of our clones are already online, so you can find them mentioning the gene name or the RefSeq in our search engine here.

Chemically competent guideline

Bacteria transformation

Chemically competent cells for plasmids and lentiviral vector amplification

High transformation efficiency is also a basic but nonetheless major point for successful cloning. GCI-5alpha are a top choice for standard plasmids, and for the lentiviral vectors, GCI-L3 are recommended.

The delivery of the construct into Mammalian

Most of the cloning vectors aim for expression into Mammalian.

Thus, the delivery efficiency of the construct should be taken in consideration as a major point. That’s why I offer these short guidelines, aimed at helping you go further. My recommendations are based on the idea that high transfection efficiency doesn’t mean a high-cost transfection reagent. Efficiency can be also low-cost.

I would suggest the PolyJet transfection reagent or the EndoFectin Max for their broad range covering many cell types.

For HEK and protein production, the LipoD293 would be an excellent choice.

Pre-optimised transfection reagents for a specific cell type is also a means to save costs and time.

Any questions about these kits, or which solutions to choose for your research? Ask me through the form below, I’ll be pleased to provide suggestions!

Dimitri Szymczak, PhD
Written by Dimitri Szymczak, PhD
Dimitri Szymczak is a Technical Support Specialist and Product Manager at tebu-bio, and a fan of capoeira in his spare time.