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.
The Fast-Fusion reveals high performances, with almost 100% cloning efficiency, on various size of inserts from 500pb to 11kpb.
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).
Gateway-based system
The EZShuttle™ LR Recombination Cloning System has the same principle as the Gateway® technology, based on recombination with Phage lambda.
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.
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.
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.
There are more than 100 pReceiver vectors corresponding to several tag possibilities and selection agents.
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.
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
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!
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