What is TA cloning kit?

The TA Cloning™ Kit uses the pCR™2.1 cloning vector and ExpressLink™ T4 DNA Ligase to generate a ligation product in a fifteen-minute, room-temperature ligation step. Reactions typically yield >80% recombinants containing inserts.

What is TA cloning vector?

TA cloning is one of the simplest and most efficient methods for the cloning of PCR products. The procedure exploits the terminal transferase activity of certain thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase.

What is TOPO TA cloning?

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.

Does TA cloning use restriction enzymes?

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.

Why is TA cloning done?

The TA cloning vector was designed so that when linearized it has single 5′ thymidine overhangs at each end. The PCR product can be ligated into this vector without the need for special restriction enzyme sites. Taq polymerase generates single 3′ A overhangs with its terminal transferase activity.

Why is TA cloning performed?

The same TA cloning vector can be used to clone any segment of PCR amplified DNA, and does not require the researcher to cut and purify the complementary vector as is done for restriction enzyme cloning. This procedure is especially useful when convenient restriction sites are not available.

How fast does Taq polymerase work?

Under conditions of moderate enzyme excess, the polymerase extends a primer at a rate of approximately 50 nucleotides per second at 70 ° C; however, rates as high as 180 nucleotides per second are possible with a very large excess of enzyme compared with template molecules (R. D. Abramson, unpublished data).

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.

How do you blunt end a ligation?

Some tips for taming blunt-end ligations

1. Tip 1: Increase concentrations of insert and ligase.
2. Tip 2: Perform the reaction in two steps.
3. Tip 3: Use longer incubation times.
4. Tip 4: Take care of how you produce the blunt ends.
5. Tip 5: Dephosphorylate the vector.
6. Tip 6: … and phosphorylate the insert.

What is the correct time for carrying out the alkaline phosphatase treatment?

4. What is the correct time for carrying out the alkaline phosphatase treatment? Explanation: The alkaline phosphatase treatment is carried out after the cutting of DNA has been done but before the mixing of insert DNA and vector has been done.

Does Taq polymerase require a primer?

Like other DNA polymerases, Taq polymerase can only make DNA if it’s given a primer, a short sequence of nucleotides that provides a starting point for DNA synthesis.

What is special about Taq polymerase?

Also, Taq DNA Polymerase is the standard for routine PCR. It is “special” because it comes from the bacterium Thermus aquaticus, which lives in hot springs. So it is thermostable even at high temperatures, while other polymerases (eg E. coli) are not.

What is end repair?

The End-Repair mix converts DNA containing damaged or incompatible 5′- and/or 3′-protruding ends to 5′- phosphorylated, blunt-ended DNA. This high-concentration formulation of the End-Repair Mix is compatible with applications requiring >1 microgram of DNA to be prepared for blunt-end ligation.

What is overhang PCR?

Overhang PCR is a technique that utilizes the intrinsic fidelity of the 3′ end of primers for a specific sequence to enable you to add on more sequence to the 5′ end (see Figure 1). This allows you to use PCR to amplify a sequence whilst adding nucleotides to either the 5′ or 3′ ends of the sequence.

What is meant by 5 overhang?

5′ overhang- Restriction enzymes that cleave the DNA asymmetrically leave several single stranded bases. If the single-stranded bases end with a 5′ phosphate, the enzyme is said to leave a 5′ overhang.

What is a 3 overhang?

An overhang is single stranded DNA at the very end of double stranded DNA. This can happen on either strand and if it is on the 5′ end of a strand, it would be a 5′ overhang and if it is on the 3′ end of a strand, it would be a 3′ overhang.