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.

What are the advantages of TA cloning?

The two chief advantages of TA cloning are speed and lack of reliance on restriction enzymes. The major disadvantage is an inability to clone directionally. For this reason, it is important to pick and analyze several transformed clones when a particular orientation of the amplified fragment is required.

What is TOPO cloning reaction?

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.

Is TOPO cloning directional?

In practise TOPO-cloning is not directional, so the insert is equally likely to ligate in both directions. However, in Geneious only the current orientation (defined by the orientations of documents in the sequence viewer) will be ligated into the vector.

What is TA plasmid?

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.

What enzyme is used in TOPO cloning?

enzyme DNA topoisomerase I
The key to TOPO cloning is the enzyme DNA topoisomerase I, which functions both as a restriction enzyme and as a ligase. Its biological role is to cleave and rejoin DNA during replication.

What is A tailing mix?

Tailing is an enzymatic method for adding a non-templated nucleotide to the 3′ end of a blunt, double-stranded DNA molecule. Tailing is typically done to prepare a T-vector for use in TA cloning or to A-tail a PCR product produced by a high-fidelity polymerase (not Taq) for use in TA cloning.

What is end repair and dA tailing?

CD End Repair/dA-Tailing Module for Illumina uses a one-step reaction to combine end-repair, 5′ phosphorylated, and dA-tailing to convert fragmented DNA into 5´-phosphorylated and 3´-dAtailed DNA fragments enabling direct ligation of Illumina sequencing adapters.