DNA cloning (gene cloning)Summary: Recominant DNA cloning involves joining (recombination) of two or more DNA fragments.
Content: DNA cloning DNA preparation step:
Preparation of vector and insert DNA
The most commonly used vector is plasmid. It usually contains several basic components:
- origin of replication for recombinant DNA replication in host bacteria.
- Multiple cloning site (MCS) containing unique restriction enzyme cutting sites for insert DNA.
- Selectable markers antibiotic resistance genes for positive clone screening.
- lacZ fragment in the MCS to enable IPTG induction for blue/white clony screening.
Commonly used vectors differ in their size and their capacity of cloning small (plasmid) to large (lambda phage and cosmid) DNA insert. For plasmid vector, it can efficiently replicate DNA insert up to 10kb.
Both vector and insert DNA can be digested with correspondant restriction enzymes to form compatible sticky end, or they can be blund-end treated by Klenow or T4 DNA polymerase to fill the 5' protruding ends for further blunt-end cloning. For PCR products cloning, it is very convenient to take advantage of the 3'-end A overhang added by Taq polymerase by cloning fresh PCR product into single T-overhang vectors (such as pGEM-T or TA cloning vectors).
Keywords: DNA cloning
Last update: 2007-03-09 16:01:29 |
