How do you clone a TA?
The TA cloning procedure begins by producing the insert in a PCR reaction using Taq polymerase, which adds a single A onto the ends of the PCR product (Fig. 7.04). Next, the PCR products are mixed with a vector that has complementary 3′ deoxythymidine (T) overhang. DNA ligase is added to connect the vector and insert.
Is TA cloning directional?
TA cloning is one of the simplest and most efficient methods for the cloning of PCR products. Directional cloning is made possible by appropriate hemi-phosphorylation of both the T-vectors and the inserts. With a single T-vector at hand, any DNA fragment can be cloned without compromising the cloning efficiency.
What is meant by TA cloning?
TA cloning (also known as rapid cloning or T cloning) is a subcloning technique that avoids the use of restriction enzymes and is easier and quicker than traditional subcloning. Such PCR amplified inserts are cloned into linearized vectors that have complementary 3′ thymine overhangs.
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.
How many base pairs are in pBR322?
4361 base pairs
pBR322 is 4361 base pairs in length and has two antibiotic resistance genes – the gene bla encoding the ampicillin resistance (AmpR) protein, and the gene tetA encoding the tetracycline resistance (TetR) protein.
How does topoisomerase work in cloning?
The key to TOPO cloning is the enzyme DNA topoisomerase I, which functions both as a restriction enzyme and as a ligase. It cleaves one DNA strand, enabling the DNA to unwind. The enzyme then relegates the ends of the cleaved strand and releases itself from the DNA.
