Since 2000, I generated quite a numbers of plasmids inserted with GOI (gene of interest).
During my master’s course,
The first project of cloning was to generate the human CD1d molecule-inserted plasmid that will be expressed in mammalian cells and set up high-expressed CD1d NKT.
Another cloning project was making a reference vector that has several cytokine fragments for PCRs for expression comparison among different cytokines.
During my Ph.D. course,
My project included generating quite a lot of in situ probes. I generated probe vectors that usually work for several species—rats, mice, and humans—even though I used them for only rat tissues.
Another project was to generate mammalian cell expression vectors for gene expression studies in cells and animals. Our group used liporfectamine, electroporation, and lentivirus for the functional studies in vitro and in vivo. I also generated siRNA fragments and dominant negative vectors to knock down gene expression. To generate antibodies, the first step I did was also to generate several different bacterial expression vectors that cover the pan, extracellular domain, and each isoform’s part.
Another project was to generate different promoter fragment-expressed vectors for luciferase assays. I generated a lot of mutagenetized promoter fragment vectors to find which promoter regions are critical.
During my post-doc stages, from time to time, whenever I felt like I needed something, I added vectors in need.
I switched from siRNA fragments to the shRNA lenti vector system and/or the Crisper/Cas9 system to knockdown or knockout genes.
I do not know when synthesized nucleotides started supporting cloning procedures. around 2010, we still used synthesized nucleotide fragments primarily for PCR probes, siRNA fragments, or mutagenesis fragments, but around 2020, I started ordering long-length synthesized fragments. That method really supports codon optimization as well.
Nowadays, people, including me, use SnapGene and other software to generate vector maps, but in 2000, I remember that we used MacVector. In or before 2000, the reason that laboratories had Macs was to use MacVector and FACS analysis software.
For cloning,
- Choose the proper vectors and check the vector maps.
- If you need to change or update the vector backbone, plan together.
- Design the final vector and set up cloning strategies.
- Order synthesized fragments and prepare backbone fragments, and other fragments needed.
- Anneal, select, screen, and confirm the sequences.
Which one is your choice: Snapgene vs. MacVector?
They are almost the same; just use the one that the company or academic lab provides!! I used MacVector, but recently I used Sanpgene. I feel they are similar without a big difference.
Which one is your choice: IDT vs. ThermoFisher or Life Technologies Gene Art for Synthesized Fragments?
You could check other companies as well, like mine, but the best choice was Life Technologies for me and my projects.
Sometimes, some fragments and others fail, but Thermo Gene Art almost succeeded.
When comparing IDT and Fisher, Fisher supports highly difficult nucleotides.
For codon optimization, please check different companies, including IDT, ThermoFisher, Nvoprolabs, etc. For the best optimization, your company might want its own program.
Which kits are yours to assemble several fragments?
NEB HiFi DNA Assembly vs. GeneArt Seamless, Gibson HiFi, EX, etc.
Generally, NEB HiFi could be comfortable with room-temperature reactions and fine up to 12 fragments.
GeneArt EX assembles up to 15 fragments.
Up to 6 to 7 fragments, both of them worked very well in my tubes.
Just check the manual and test them!
For mutagenesis, the most common method is to just use PCR using fragments; I never used commercial mutagenesis kits. There are no reasons to use them.
Maybe you heard about T vector cloning. I think there is no reason to use T vector cloning system any more, but a long time ago, in or before 2000, we made a T vector system in the lab just using a PCR machine, a blunt-ended vector, and dTTP. Within 2 hours, we could make a T vector backbone for the next step.
Who believes that in the old days of laboratories, we generated lab-made taqs, lab-made pfu, lab-made Lama phage DNA ladders, and more for our own experiments? Who believes we made PCR buffers as well!!
If you need consider Patent application for your design or GOI for your team and your company, you might use Patsnap !
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