What is Quick ligase?

The Quick Ligation™ Kit enables ligation of cohesive end or blunt end DNA fragments in 5 minutes at room temperature. ( 25°C ) Fast – 5 minutes for cohesive or blunt ends. Convenient – ligation performed at room temperature. Flexible – suitable for all common ligation reactions.

What are the steps of a ligation reaction?

The three steps to form a new phosphodiester bond during ligation are: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing.

What is a ligation reaction?

This reaction, called ligation, is performed by the T4 DNA ligase enzyme. The DNA ligase catalyzes the formation of covalent phosphodiester linkages, which permanently join the nucleotides together. The majority of ligation reactions involve DNA fragments that have been generated by restriction enzyme digestion.

How long does a ligation reaction take?

Typical ligation reactions use 100–200ng of vector DNA. 2. Incubate the reaction at: room temperature for 3 hours, or 4°C overnight, or 15°C for 4–18 hours.

How does quick ligase work?

The Quick Ligation Kit enables ligation of cohesive end or blunt end DNA fragments in 5 minutes at room temperature. 1. Combine 50 ng of vector with a 3-fold molar excess of insert. Adjust volume to 10 µl with dH2O.

How can I increase my ligation efficiency?

Include polyethylene glycol (PEG) PEG is a hydrophobic molecule that takes up space in the reaction, effectively increasing the concentration of the aqueous reaction components e.g. DNA, ATP and ligase. Adding PEG (e.g. PEG 8000) to a final concentration of 5-15% may increase ligation efficiency.

How do you know if ligation worked?

You may run the ligation product on the gel to see if it worked. You should see multiple bands of higher molecular weight than your empty vector, as well as a band of the same size of the insert, since you should be using an excess of insert. Also, before ligating you should smell the ligase buffer.

How do you increase ligation efficiency?

PEG is a hydrophobic molecule that takes up space in the reaction, effectively increasing the concentration of the aqueous reaction components e.g. DNA, ATP and ligase. Adding PEG (e.g. PEG 8000) to a final concentration of 5-15% may increase ligation efficiency.

How do you stop a ligation reaction?

This can generally be controlled by decreasing the enzyme concentration and/or time of the reaction. Enzyme concentration, glycerol concentration, high salt, and impurities can contribute to sub-optimal reaction conditions. Incomplete inactivation of the restriction enzyme can cause ligation to fail.

What is the function of PEG in ligation?

Polyethylene glycol or PEG, is included in ligation reactions to increase the reaction rate and the overall yield. PEG does this by being a macromolecular crowding agent, which ends up increasing the effective concentration of both the ligase and the DNA substrate in the tube.

How do you use a quick CIP?

1. The phosphatase can be added directly into the digestion reaction during or after DNA digestion.
2. Add 1 μl of Quick CIP for every 1 pmol of DNA ends (about 1 μg of a 3 kb plasmid) and incubate at 37°C for 10 minutes.
3. Quick CIP is active in all NEB restriction enzyme buffers.