Why is subcloning important?

Subcloning is a basic procedure in molecular biology for transfer of DNA inserts from one vector to another to gain functionality to study the sequence of interest.

What is TOPO cloning used for?

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 is the advantage of using two restriction enzymes?

The use of 2 different enzymes makes self ligation of the vector impossible and makes the insertion unidirectional. Whereas in the case of single digest, selfligation occurs and insertion may occur in both ways. Overall the use of 2 RE increases the probability to get the right construct.

What is the difference between subcloning and cloning?

Cloning is the procedure which produces genetically identical organisms or cells. Subcloning is a procedure of moving a gene of interest from one vector to another vector to see the expression of the gene to gain the desired functionality of the gene.

What is subcloning vs molecular cloning?

The main difference between cloning and subcloning is that cloning is the production of clones of organisms or copies of cells or DNA fragments whereas subcloning is a technique used to move a particular DNA sequence from a parent vector to a destination vector.

What is the principle of TA cloning?

The TA cloning method takes advantage of the terminal transferase activity of some DNA polymerases such as Taq polymerase. This enzyme adds a single, 3′-A overhang to each end of the PCR product. This makes it possible to clone this PCR product directly into a linearized cloning vector with single, 3′-T overhangs.

Why are Type 2 restriction enzymes used for cloning?

Type II restriction enzymes are the familiar ones used for everyday molecular biology applications such as gene cloning and DNA fragmentation and analysis. These enzymes cleave DNA at fixed positions with respect to their recognition sequence, creating reproducible fragments and distinct gel electrophoresis patterns.

Why is it important to perform a restriction digest after cloning?

Digestion Set up restriction digests for your insert (or donor plasmid) and plasmid backbone. Because you lose some DNA during the gel purification step, it is important to digest plenty of starting material.

What is A TA cloning vector?

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.

Is pBR322 A shuttle vector?

A series of shuttle vectors for Bacillus subtilis and Escherichia coli was developed. These are derived from one basic construct composed of parts of the Gram+ plasmid pUB110 and the Gram- plasmid pBR322. They contain multiple cloning sites flanked by transcriptional terminators.