What do you need to know to start cloning?
Plasmids are circular extrachromosomal DNA molecules of bacteria that can be used in genetic engineering for cloning or expression in both prokaryotic and eukaryotic systems. Essential elements of any plasmid include a bacterial origin of replication and an antibiotic resistance gene. Below is a brief description of the main structural and functional elements of plasmids:
After a sequence is inserted into a plasmid, its initial amplification is carried out in a prokaryotic system - in the bacterium Escherichia coli (E. coli). This is a Gram-negative bacterium that is a natural symbiont of the intestines of humans and animals. In cloning, laboratory strains of E. coli are used; these carry a number of mutations that ensure the stability of plasmids and cloned sequences. Below are the most common laboratory E. coli strains and the characteristics of their genotypes:
As selectable markers, different antibiotic resistance genes can be used depending on the objectives of the experiment. Some antibiotics used in genetic engineering are listed in the table below:
To read plasmid maps and sequences, it is also important to know the letter abbreviations for nucleotides and amino acids. We provide them in the tables below:
NUCLEIC ACIDS
AMINO ACIDS
Various epitopes can also be used in plasmids as part of the expressed protein. Commercial antibodies may be available against these epitopes, or they may be useful for chromatography and adsorption. The most commonly encountered epitopes are listed below:
Materials from Addgene.org were used.
For more detailed information, please contact DUOX BIOTECHNOLOGIES specialists, and we will help you design the genetic constructs needed for your work.Использованы материалы addgene.org