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Bacterial cloning is a crucial technique in molecular biology, allowing for the replication of specific DNA sequences by creating numerous identical copies within bacterial cells. This method, especially applicable to PCR products, ensures high accuracy in DNA amplification. By utilizing plasmid vectors and techniques such as electroporation, scientists can clone and express protein-coding sequences in E. coli, enabling the production of desired proteins. Understanding Taq polymerase's unique properties, including its characteristic A overhang, is vital for effective cloning strategies.
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Bacterial cloning Especially of PCR product DNA
Cloning • Cloning is the way in which we can take a single molecule, and make lots of bacterial cells that contain an identical molecule. • These cells are clones, hence the name • This used to be the only way to amplify DNA. It is still by far the most accurate.
Cloning PCR products • When we amplify DNA using PCR, it is often necessary to “clone” this DNA • We do this in order to replicate it without errors • Also, by cloning a protein coding sequence into E. coli, we can then produce the protein in the bacterium.
Taq polymerase leaves an “A” overhang • Taq is the thermostable DNA polymerase from Thermus aquaticus we used for PCR. • When Taq synthesizes a new strand, it always puts an extra “A” at the end • This can be useful, but note: other polymerases do not do this, only Taq polymerase
When the electric field is applied, • the ions move according to their • charge. • ii) Pathways are formed across the • cell membrane allowing DNA • to enter. • iii) When the electric field is • deactivated, the membrane heals.
