How does topo TA cloning work?
How does topo TA cloning work?
Topoisomerase based cloning (TOPO cloning) is a DNA cloning method that does not use restriction enzymes or ligase, and requires no post-PCR procedures. Sounds easy right? The technique relies on the basic ability of complementary basepairs adenine (A) and thymine (T) to hybridize and form hydrogen bonds.
What is a TOPO cloning kit?
The TOPO™ TA Cloning™ Kits for Sequencing provide a highly efficient, 5-minute, one-step cloning strategy (‘TOPO™ Cloning’) for the direct insertion of Taq polymerase–amplified PCR products into a plasmid vector for sequencing.
What is a TOPO cloning vector?
TOPO cloning is a molecular biology technique in which DNA fragments are cloned into specific vectors without the requirement for DNA ligases. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3′-end of the PCR products.
Is TA cloning directional?
Although TA cloning is widely used to clone unmodified PCR (polymerase chain reaction) products, a major disadvantage of this technique is that cloning is not directional.
How does blunt end ligation work?
Blunt-end cloning involves the ligation of DNA fragments – usually between a plasmid vector and an insert – whose terminal ends are not “sticky”. Performing these ligations is notoriously difficult, particularly with large DNA fragments.
How is PCR used in cloning?
In its simplest form, PCR based cloning is about making a copy of a piece of DNA and at the same time adding restriction sites to the ends of that piece of DNA so that it can be easily cloned into a plasmid of interest.
Why is it called TA cloning?
The technique relies on the ability of adenine (A) and thymine (T) (complementary basepairs) on different DNA fragments to hybridize and, in the presence of ligase, become ligated together.
